Negative selection and stringency modulation in continuous evolution systems

ABSTRACT

Strategies, systems, methods, reagents, and kits for phage-assisted continuous evolution are provided herein. These include strategies, systems, methods, reagents, and kits allowing for stringency modulation to evolve weakly active or inactive biomolecule variants, negative selection of undesired properties, and/or positive selection of desired properties.

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. §371 ofinternational PCT application, PCT/US2015/012022, filed Jan. 20, 2015,which claims priority under 35 U.S.C. §119(e) to U.S. provisional patentapplication, U.S. Ser. No. 61/929,378, filed Jan. 20, 2014, each ofwhich is incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under grant numberHR0011-11-2-0003 awarded by U.S. Defense Advanced Research ProjectsAgency (DARPA) and under grant number N66001-12-C-4207 awarded by U.S.Space and Naval Warfare Systems Center (SPAWAR). The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

Proteins and nucleic acids employ only a small fraction of the availablefunctionality of these types of molecules. There is currentlyconsiderable interest in modifying proteins and nucleic acids todiversify their functionality. Molecular evolution efforts include invitro diversification of a starting molecule into related variants fromwhich desired molecules are chosen. Methods used to generate diversityin nucleic acid and protein libraries include whole genome mutagenesis(Hart et al., Amer. Chem. Soc. (1999), 121:9887-9888), random cassettemutagenesis (Reidhaar-Olson et al., Meth. Enzymol. (1991), 208:564-86),error-prone PCR (Caldwell et al., PCR Methods Applic. (1992), 2: 28-33)and DNA shuffling using homologous recombination (Stemmer, Nature(1994), 370:389-391). After diversification, molecules with novel orenhanced properties can be selected.

Conventional directed evolution involves discrete cycles of mutagenesis,transformation or in vitro expression, screening or selection, and geneharvesting and manipulation.^(1,2) In contrast, evolution in natureoccurs in a continuous, asynchronous format in which mutation,selection, and replication occur simultaneously. Although successfulevolution is strongly dependent on the total number of rounds performed,the labor- and time-intensive nature of discrete directed evolutioncycles limit many laboratory evolution efforts to a modest number ofrounds.

In contrast, continuous directed evolution has the potential todramatically enhance the effectiveness of directed evolution efforts byenabling an enormous number of rounds of evolution to take place in asingle experiment with minimal researcher time or effort. Whilelaboratories have explored various aspects of continuous evolution, nogeneralizable, continuous directed evolution system has been reported.In a landmark experiment, Joyce and co-workers engineered a ribozymeself-replication cycle in vitro and used this cycle to continuouslyevolve a ribozyme with RNA ligase activity (Wright, M. C. & Joyce, G. F.(1997) Science 276, 614-617). However, the foregoing example ofcontinuous directed evolution cannot be easily adapted to evolve otherbiomolecules.

Continuous directed evolution minimally requires (i) continuousmutagenesis of the gene(s) of interest, and (ii) continuous selectivereplication of genes encoding molecules with a desired (on-target)activity. Several groups have developed methods to achieve continuous orrapid non-continuous cycles of mutagenesis. For example, Church andcoworkers recently developed multiplex automated genome engineering(MAGE), a system capable of generating targeted diversity in E. colithrough automated cycles of transformation and recombination (Wang, H.H. et al. (2009) Nature 460, 894-898). While these advances are capableof very efficiently creating gene libraries, they have not been linkedto a rapid and general continuous selection and consequently have notenabled continuous directed evolution.

SUMMARY OF THE INVENTION

Many biomolecules, such as nucleic acids and proteins, have beengenerated in the laboratory with desired properties using continuousdirected evolution. Continuous directed evolution strategies have beendescribed in, for example, International Patent Application No.PCT/US2009/056194 and PCT/US2011/066747 and elsewhere^(1,6,7), each ofwhich is hereby incorporated by reference in its entirety. Describedherein are strategies, systems, methods, reagents, and kits to expandthe scope and capabilities of continuous directed evolution and addressthe needs of previously described laboratory evolution experiments.Generally, the continuous directed evolution of gene-encoded moleculescan be linked to protein production in a host cell, for example, in anE. coli cell. Methods have been described for evolving a gene ofinterest by linking an activity of a molecule encoded by the gene ofinterest to the transfer of the gene of interest from cell to cell. Theevolving gene of interest is transferred from host cell to host cellthrough a modified viral life cycle in a manner that is dependent on theactivity of the molecule of interest. The gene of interest is replicatedand mutated in a flow of host cells. The desired function of the gene ofinterest drives expression of a gene in the host cells that is essentialfor transfer of the gene from one cell to another, thus providing aselective advantage for those viral vectors in which the gene ofinterest has acquired a relevant gain-of-function mutation. Dozens ofcycles of viral replication, mutation, and selection can occur in asingle day of directed continuous evolution without human intervention.

In some embodiments, the continuous directed evolution method isphage-assisted continuous evolution (PACE). During PACE, a population ofbacteriophage, each encoding a library member of an evolving gene,selectively replicates in a “lagoon” of continuously replenished hostcells in a manner that depends on an activity of interest (FIGS. 1a and1b ).¹ A gene encoding a phage protein required for the production ofinfectious phage particles is under the control of a conditionalpromoter the activity of which depends on a gene product encoded by thegene of interest. Since phage life cycles are generally short (e.g., inthe range of 10-20 minutes), many phage life cycles can occur in asingle day of directed continuous evolution without human intervention.

Previous efforts of continuous directed evolution, such as PACE,utilized relatively high selection stringency to evolve biomoleculeswith desired properties such as broadened specificity. Improvementsdescribed herein include the ability to modulate the selectionstringency to also allow weakly active or inactive biomolecule variantsto access favorable mutations that enable their propagation undersubsequent higher selection/stringency conditions. In addition,strategies included herein are for explicit negative selection againstan undesired (off-target) property, which allows evolution ofbiomolecules with one or more altered properties (e.g., alteredactivity, specificity, stability and/or enantioselectivity). Thus, thecombination of stringency modulation and negative selection enables thecontinuous directed evolution of biomolecules with a broader scope ofevolved properties such as altered and highly specific new activities.

The continuous directed evolution methods herein utilize either or bothof the following: 1) general modulation of selection stringency toenable otherwise inaccessible properties of biomolecules to be evolveddirectly from weakly active or inactive starting genetic librariesthrough a period of evolutionary drift that is independent of theactivity being evolved; and/or 2) general negative selection to providefor continuous counter-selection against undesired properties ofbiomolecule variants.

In one aspect, general modulation of selection stringency to enableotherwise inaccessible properties of biomolecules to be evolved directlyfrom weakly active or inactive starting genes through a period ofevolutionary drift is controlled using a concentration of a smallmolecule in a manner independent of the activity being evolved. In someembodiments, the continuous directed evolution methods are performedusing reduced or low selection stringency conditions. In someembodiments, the continuous directed evolution methods are performedusing higher selection stringency conditions. In other embodiments, thecontinuous directed evolution methods are performed using conditionswhich combine reduced and higher selection stringency conditions. Insome embodiments, low selection stringency conditions are used followedby high selection stringency conditions. In some embodiments, themethods optionally further use reduce or relatively more reduceselection stringency conditions or further use high or relatively higherselection stringency. In some embodiments, a set of conditions is useduntil an evolved product with certain properties is achieved or thelevel of a certain evolved product has been stabilized. In someembodiments, the method can optionally use one or more different sets ofconditions to achieve the final desired evolved product or intermediateproduct.

In some embodiments, provided are methods for viral-assisted evolutionof a gene product, the method comprising introducing a selection viralvector (such as a phagemid) comprising a gene to be evolved into a flowof host cells through a lagoon, wherein the host cells comprise viralgenes required to package the selection viral vector into infectiousviral particles, wherein at least one gene required to package theselection viral vector into infectious viral particles is expressed inresponse to a desired property of a gene product encoded by the gene tobe evolved or an evolution product thereof; further wherein the hostcells comprise a second copy of viral genes required to package theselection viral vector into infectious viral particles, wherein at leastone gene required to package the selection viral vector into infectiousviral particles is expressed in response to the concentration of a smallmolecule.

In some embodiments, provided are methods for viral-assisted evolutionof a gene product, the method comprising introducing a selection viralvector (e.g., a selection phagemid) comprising a gene to be evolved intoa flow of host cells through a lagoon, wherein the host cells contain alow selection stringency plasmid and a high selection stringencyplasmid, wherein the concentration of a small molecule is used tocontrol the level of selection stringency that dominates. The lowselection stringency plasmid contains viral genes required to packagethe selection viral vector into infectious viral particles, wherein theviral gene is controlled by a small-molecule inducible promoter to allowevolutionary drift to occur. The high selection stringency plasmidcontains viral genes required to package the selection viral vector intoinfectious viral particles, wherein the viral gene is controlled by anactivity-dependent promoter, wherein the activity is the activity of thegene product being evolved. In some embodiments, the selectionstringency is inversely proportional to the concentration of the smallmolecule. In some embodiments, the low selection stringency plasmid isdominant over the high selection stringency plasmid using a saturatingor high concentration of the small molecule. In some embodiments, thehigh selection stringency plasmid is dominant over the low selectionstringency plasmid using zero or a low concentration of the smallmolecule. In some embodiments, low selection stringency conditionsallows evolutionary drift to occur, thereby allowing the propagation ofweakly active or inactive starting genes. In some embodiments, highselection stringency enables the evolution of active starting genes.

One aspect provided herein is a drift promoter that is induced by thecombination of prior viral infection and the presence of a smallmolecule inducer. The drift promoter is found on a drift plasmid in thehost cell and enables evolutionary drift to occur. The drift promoter isused to support propagation of the selection viral vector by drivingexpression of a viral gene required to package the selection viralvector into infectious viral particles in a manner that is independentof the activity being evolved. In some embodiments, the drift promoterdrives expression of a gene that encodes the pIII protein. In someembodiments, the low selection stringency plasmid contains the driftpromoter. In some embodiments, the drift promoter reduces the hostcell's resistance to viral infection. In some embodiments, the driftpromoter supports activity-independent viral propagation (i.e., supportviral propagation in a manner that is independent of the desiredevolving activity from the selection viral vector). In some embodiments,the prior viral infection is a prior phage infection. In someembodiments, the viral propagation is phage propagation. In someembodiments, the small molecule inducer is a tetracycline ortetracycline analog. In some embodiments, the small molecule inducer isthe tetracycline analog, anhydrotetracycline (ATc). In one embodiment,the small molecule is doxycycline. In one embodiment, the drift promoteris a combination of promoters comprising a tetracycline-induciblepromoter (P_(tet)), which natively drives expression of the TetRrepressor and TetA, the protein that pumps tetracycline out of the cell.In one embodiment, the drift promoter is a combination of promoterscomprising an E. coli phage shock promoter (P_(psp)). In oneembodiments, the drift promoter is P_(psp-tet), which is a combinationof a E. coli phage shock promoter (P_(psp)) with TetR operatorsinstalled at the position adjacent to the +1 transcription initiationsite.

One aspect provided herein is a vector system in a host cell wherein oneof the vectors is controlled by a small molecule inducer to evolveproducts from either the active or weakly active/inactive starting geneor genetic libraries cloned into the viral genome of a selection viralvector. In some embodiments, a vector system in a host cell contains avector that is a drift plasmid encoding a viral gene required to packagethe selection viral vector into infectious viral particles, wherein theviral gene on the drift plasmid is under the control of a host celldrift promoter that is induced by the combination of prior viralinfection and the presence of a small molecule inducer. In someembodiments, induction of the drift plasmid allows evolved products tobe generated from weakly active or inactive starting genetic libraries.In one embodiment, host cell drift promoter comprises the P_(psp-tet)promoter. In some embodiments, the drift plasmid contains a mutagenesiscassette under the control of a second small molecule induciblepromoter. In some embodiments, the second small molecule induciblepromoter is induced by arabinose.

In one aspect, the vector system further includes another vector that isan accessory plasmid encoding a viral gene required to package theselection viral vector into infectious viral particles, wherein theviral gene on the accessory plasmid is under the control of the desiredevolving activity, wherein the desired evolving activity is producedfrom a selection viral vector.

In another aspect, a general negative selection against an undesiredproperty is used in the continuous evolution system described. In someembodiments, the negative selection alters the activity, specificity,stability, or enantioselectivity of a biomolecule such as an enzyme. Insome embodiments, the negative selection alters the specificity of abiomolecule such as an enzyme. In some embodiments, the evolved propertyresulting from negative selection or from both positive and negativeselection is altered and/or broadened biomolecule specificity. Forexample, negative selection can be used to select an enzyme that isspecific for a both the natural substrate and a non-natural substrate orselect an enzyme that is specific for the non-natural substrate over thenatural substrate. In some embodiments, negative selection is used toevolve the gene of interest. In some embodiments, a combination ofselection stringency modulation and negative selection is used togenerate an evolved product. In certain embodiments, the continuousevolution strategies utilize both negative and positive selection toevolve the gene of interest.

In another aspect, general negative selection to enable continuouscounter-selection against undesired properties of biomolecules isaccomplished using a dominant negative mutant gene of a viral generequired to package the selection viral vector into infectious viralparticles, wherein the expression of the dominant negative mutant geneis controlled using, for example, a promoter inducible by a smallmolecule. In another embodiment, the expression of the dominant negativemutant gene is controlled using a promoter comprising an undesiredDNA-binding site for the gene product. In some embodiments, the dominantnegative mutant gene is expressed in response to an undesired activityof the gene being evolved or a product of the gene being evolved. Insome embodiments, the dominant negative mutant gene, or expressionproduct thereof, decreases or abolishes the ability of the selectionviral vector to generate infectious viral particles. In someembodiments, the dominant negative mutant gene prevents or inhibits thedetachment of nascent phage from the host cell membrane.

The above embodiments are useful in the various continuous directedevolution systems and methods described herein and in InternationalPatent Applications, No. PCT/US2009/056194 and No. PCT/US2011/066747. Insome of the above embodiments, the virus is a phage or phagemid.

In some embodiments, the viral-assisted continuous evolution isphage-assisted continuous evolution (PACE), further described herein. Insome embodiments, the selection viral vector is a selection phage orphagemid (SP). In some embodiments, the selection phagemid is an M13phagemid. In some embodiments, the viral genes required to package theselection viral vector into infectious viral particles is a phage generequired to package the selection phage into infectious phage particles,wherein at least one phage gene required to package the selection phageinto phage particles is expressed in response to a desired property of agene product encoded by the gene to be evolved or an evolution productthereof. In some embodiments, the host cells comprises a second copy ofa phage gene required to package the selection phage vector intoinfectious phage particles, wherein at least one gene required topackage the selection phage into phage particles is expressed inresponse to the concentration of a small molecule. In some embodiments,the phage gene required to package the selection phage vector is one ormore genes selected from the gene which encodes the pII protein, thepIII protein, or the pVI protein. In some embodiments, the gene is oneor more genes selected from gene II, gene III, or gene VI. In someembodiments, the phage gene required to package the selection phagevector into infectious phage particles is a gene which encodes the pIIIprotein. In some embodiments, the gene is gene III. In some embodiments,the dominant negative mutant gene encodes a dominant negative mutantprotein of the pII protein (i.e., pII-neg protein), pIII protein (i.e,pIII-neg protein), or the pVI protein (i.e., pVI-neg protein). In someembodiments, the dominant negative mutant gene encodes a dominantnegative mutant protein of the pIII protein (i.e, pIII-neg protein). Insome embodiments, the dominant negative mutant gene is gene II-neg, geneIII-neg, or gene VI-neg In some embodiments, the dominant negativemutant gene is gene III-neg.

In some embodiments, the dominant negative pIII protein comprises amutant C domain, wherein the amino acids in the C-domain have beentruncated. In some embodiments, the dominant negative pIII proteincomprises an N-C83 domain, which has an internal deletion of 70 aminoacids (i.e., amino acids 1-70) from the C-terminal domain of the pIIIprotein.¹⁶

Some aspects of this disclosure provide negative viral selectionconstructs. Such constructs are useful in the vector systems describedherein. The negative viral selection constructs can be used withpositive viral selection constructs. In some embodiments, the negativeand positive viral selection constructs are located on differentplasmids. In some embodiments, the negative viral selection construct isa negative phage selection construct. In some embodiments, the positiveviral selection construct is a positive phage selection construct. Insome embodiments, the negative selection construct comprises a nucleicacid encoding a dominant negative mutant of a viral gene product thatdecreases or abolishes the ability of the selection phagemids togenerate infectious phage particles and a promoter driving expression ofthe encoded dominant negative mutant gene product, wherein the promotercomprises a DNA target site for an undesired property of a gene to beevolved. In some embodiments, the stringency of the negative and/orpositive selection can be modified by decreasing or increasing thestrength of a ribosome binding site upstream of the phage gene requiredto package the selection phage vector (such as a gene encoding pIII)and/or using a low or high copy number plasmid.

Another aspect provided herein is a vector system in one or more hostcells comprising negative and positive viral selection constructs. Insome embodiments, one or both the viral genes are controlled by smallmolecule inducers to evolve products toward the desired activity andaway from the undesired activity. In some embodiments, one of the viralgenes is driven by a promoter induced by the desired activity. In someembodiments, one viral gene is driven by a promoter induced by thedesired activity and another viral gene is controlled by small moleculeinducers. In some embodiments, the negative viral selection construct isdriven by a promoter induced by a small molecule and the positive viralselection construct is driven by a promoter induced by the desiredactivity.

In a further aspect provided herein, a gene to be evolved encodes aDNA-binding gene product (e.g., polymerases, transcription factors,nucleases, or methylases). In some embodiments, expression of the generequired to package the selection phagemid into infectious particles isdriven by a promoter comprising a desired DNA binding site for theDNA-binding gene product. In some embodiments, the DNA-binding geneproduct is a T7 RNA polymerase (T7 RNAP). In some embodiments, thepromoter comprising a desired DNA binding site for the gene product is aT7 promoter (P_(T7)) or a T3 promoter (P_(T3)). In some embodiments, theevolved product is an evolved T7 RNA polymerase which has activity onthe P_(T3). In some embodiments the evolved product is a T7 RNApolymerase which is specific for the P_(T3) over the P_(T7). In someembodiments, the specificity for the evolved product for the non-nativeDNA-binding site exceeds the specificity for the product of the originalgene to be evolved for the native DNA-binding site. In one embodiment,the specificity of an evolved T7 RNA polymerase for P_(T3) over P_(T7)exceeds the specificity of a wild-type T7 RNA polymerase for P_(T7) overP_(T3). In some embodiments, the T7 RNA polymerase is at least about1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 80-fold, 100-fold,500-fold, 800-fold, 1000-fold, 3,000-fold, 5,000-fold, 8,000-fold,10,000-fold, 12,000-fold, or 15,000-fold more specific for the P_(T3)over the P_(T7). In some embodiments, the T7 RNA polymerase is at leastabout 1.5-fold to 10-fold, 10-fold to 50-fold, 50-fold to 80-fold,80-fold to 100-fold, 100-fold to 300-fold, 300-fold to 500-fold,500-fold to 800-fold, 1000-fold to 3,000-fold, 3,000-fold to 5,000-fold,5,000-fold to 8,000-fold, 8,000-fold to 10,000-fold, 10,000-fold to12,000-fold, or 12,000-fold to 15,000-fold more specific for the P_(T3)over the P_(T7).

In some embodiments, the evolved product is a T7 RNA polymerase evolvedfrom a T7 RNAP that is substrate-specific for P_(T7) rather than from aT7 RNAP that is promiscuous. In some embodiments, expression of thedominant negative mutant gene is driven by a promoter comprising anundesired DNA-binding site for the DNA-binding gene product. In someembodiments, the undesired DNA binding site is an off-target DNA bindingsite. In some embodiments, the promoter is a T3 promoter (P_(T3)).

Further, some aspects of this invention provide kits comprisingreagents, vectors, cells, software, systems, and/or apparatuses forcarrying out the methods provided herein. For example, in someembodiments, a kit for controlling the selection stringency in acontinuous directed evolution in a bacterial system is provided thatincludes a selection phage or phagemid; a drift plasmid; an accessoryplasmid; optionally, a mutagenesis plasmid and/or a mutagen; and/or ahost cell capable of producing infectious phage and amenable to phageinfection. In some embodiments, a kit is provided that comprises atwo-plasmid PACE vector system, as described in more detail elsewhereherein, for example, comprising a selection phage, a drift plasmid, anaccessory plasmid, optionally, a mutagenesis plasmid, but no helperphage. In some embodiments, the kit further contains negative and/orpositive selection constructs and optionally, a mutagenesis plasmidand/or a mutagen. In some embodiments, the kit optionally contains smallmolecule inducers. The kit typically also includes instructions for itsuse.

Other advantages, features, and uses of the invention will be apparentfrom the Detailed Description of Certain Embodiments, the Drawings, theExamples, and the Claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-E. PACE overview and development of a gene III expressioncassette that enables drift during continuous phage propagation. (a-b)During PACE, host E. coli cells continuously dilute an evolvingpopulation of filamentous bacteriophages in a fixed-volume vessel (a“lagoon”, detailed in (b)). The lagoon is continuously drained to awaste container after passing through an in-line luminescence monitorthat measures expression from a gene III-luciferase cassette on the AP.Dilution occurs faster than cell division but slower than phagereplication. Each phage carries a protein encoding gene to be evolvedinstead of a phage gene (gene III) that is required for infection. Phageencoding active variants trigger gene III expression in proportion tothe desired activity and consequently produce infectious progeny, whilephage encoding less active variants produce fewer infectious progeny andare diluted out of the lagoon. (c-e) Cells harboring the indicatedaccessory plasmids (APs) with the indicated gene III-luxAB expressioncassettes were used as recipients for phage propagation experimentsusing selection phages encoding the wild-type T7 RNA polymerase(SP-T7_(WT)). Data show representative single measurements of phageconcentrations (n=1). (c) Using the tetracycline-inducible promoter,P_(tet), to induce gene III expression with anhydrotetracycline (ATc)prior to phage infection inhibits infection and results in minimal phagepropagation and low phage titers. (d) Using P_(psp), an E. coli phageshock promoter, to express gene III only after infection takes placeresults in robust activity-independent phage propagation and high phagetiters. (e) Infection- and ATc-dependent gene III expression usingP_(psp-tet) enables robust, activity-independent propagation.

FIG. 2A-E. Characterization of candidate gene III cassettes formodulating stringency and enabling drift. (a) Architecture of P_(tet)(top; 148i2), P_(psp) (middle; 175e), and P_(psp-tet) candidates(bottom; 175g, h, k, m, and n variants). For the P_(psp-tet) candidates,the numbers shown in parentheses refer to the constructs that containthe tetR-binding sites at the positions shown by the orange bar.tetR:tetR-binding sites, pspF:pspF-binding sites, σ70:E. coli sigma 70RNAP promoter, σ54: E. coli sigma 54 RNAP promoter. (b) Architecture ofplasmids highlighted in this figure. (c) Luciferase gene expressionmeasurements of candidate drift cassettes in the presence and absence ofATc (which de-represses TetR) and IPTG (which de-represses lad andinduces pIV expression). pIV expression is used for these experiments toemulate filamentous phage infection and drive the P_(psp) response. (d)Phage production from discrete cultures containing various driftconstructs and SP-T7_(WT) in the presence and absence of ATc (to inducegene III expression) and arabinose (to induce mutagenesis). Robust phagetiters are produced regardless of drift cassette copy number ormutagenesis induction. (e) Assay for infection of recipient cellscarrying the pTet-pIII cassette. A low proportion of yellow coloniesindicates resistance to infection, as seen for recipient cells carryingthe induced pTet construct.

FIG. 3A-D. Drift cassette enables ATc-dependent, activity-independentphage propagation. Cells harboring APs with the indicated gene III-luxABexpression cassettes served as recipients for phage propagationexperiments using SP-T7_(WT). Data show representative singlemeasurements of phage concentrations (n=1). (a) Recipient cells carryinga drift plasmid (DP) and a P_(T7)-gene III AP were used to propagate amixture of SP-T7WT (wild-type T7 RNAP, high activity) and SP-T7Dead(D812G mutant T7 RNAP,¹⁴ no activity) at a ratio of 1:10² (“Mix” in(b)). (b) In the absence of ATc, SP-T7WT (WT) is rapidly enriched overthe inactive D812G mutant polymerase (“D”) and a rapid increase inluciferase signal is observed. (c) At an intermediate ATc concentration(150 ng/mL), SP-T7WT is enriched at a slower rate, concurrent with aslower rise in luciferase signal. (d) At the highest ATc concentration(400 ng/mL), SP-T7WT is not enriched and baseline luciferase signal isobserved. Upon ending ATc supplementation at t=8 h, SP-T7WT is rapidlyenriched, and the luciferase signal rapidly rises.

FIG. 4. Effect of pIII-neg candidate expression on infectious phagetiter. Gene III expression is driven by P_(tet) (induced by ATc), andthe expression of a gene III-neg candidate is driven by pLac (induced byIPTG). Constructs from left to right are pJC156a2 (C-domain), pJC156c2(C83), pJC156j2 (N-C83), pJC156m2 (N2-C83), pJC156o2 (N*-C83). Aminoacid sequences of the pIII-neg candidates are provided in FIG. 5. ATcwas used at 4 or 20 ng/mL, and IPTG was used at 0 or 2 mM.

FIG. 5. Sequence alignment of pIII-neg candidates and wild-type pIII.ClustalW2 (www.ebi.ac.uk/Tools/msa/clustalw2/) was used to align allpIII-neg candidates against full-length pIII.

FIG. 6. Dose-dependent effect of pIII-neg expression on phageproduction. A selection phage (SP) encoding a promiscuous T7 RNAPvariant (SP-T7_(Prom)) or an SP encoding a P_(T7)-specific RNAP variant(SP-T7_(Spec)) were used to infect cells harboring an AP in which P_(T7)drives gene III expression and an AP-neg in which P_(T3) drives geneIII-neg expression in a theophylline-dependent manner. Cells wereinfected using excess phage for 10 min at 37° C., centrifuged and washedto remove residual phage, and resuspended in fresh Davis rich media.Infected cells were grown in the presence of the indicatedconcentrations of theophylline. The resulting titers of progeny phageafter reaching mid-log phase are shown in the graph. SP-T7_(Prom)results in fewer progeny phage only when theophylline is added. Thetotal secreted DNA was also measured (bottom) and correlated with phageproduction. The X-axis numbers indicate the concentration oftheophylline in micromolar (μM).

FIG. 7A-D. Dominant negative pIII-neg is a potent inhibitor of phagepropagation. (a) Recipient cells carrying a P_(T7)-gene III AP and aP_(T3)-gene III-neg APneg in which the theophylline riboswitch controlsgene III-neg expression were used to propagate a 1:10⁶ mixture (“mix” in(b)) of SP-T7_(Spec) (specific for P_(T7), “spec”) and SP-T7_(Prom)(promiscuous on both P_(T7) and P_(T3), “prom”), respectively. (b) At ahigh theophylline concentration (1000 μM), the promiscuous T7 RNAP SP israpidly depleted, and the specific T7 RNAP SP quickly takes over thelagoon, concomitant with a sharp rise in luciferase signal from P_(T7).(c) At an intermediate theophylline concentration, the promiscuous T7RNAP SP slowly washes out and is gradually replaced by the specific T7RNAP SP, accompanied by a less drastic drop then gradual recovery ofluciferase signal. (d) In the absence of theophylline, the promiscuousT7 RNAP SP propagates unhindered, and the lagoon maintains the startingratio of the inoculated phage. Upon addition of high concentrations oftheophylline to this lagoon at t=12 h, a rapid washout of thepromiscuous T7 RNAP SP takes place, with a rebound in luciferase signalconsistent with specific T7 RNAP SP enrichment. In (b)-(d), data showrepresentative single measurements (n=1).

FIG. 8. Schedule for the continuous evolution of T3-specific RNAPvariants. PACE of T7 RNAP variants that recognize P_(T3) and rejectP_(T7) was performed in three contiguous stages of differing stringency,using three host cell strains carrying the combinations of plasmidsshown (bottom). Arabinose was added to the lagoons at all timepoints toinduce high levels of mutagenesis from the drift plasmid (DP) ormutagenesis plasmid (MP). The P_(T3) and P_(T7) bars conceptuallyrepresent amounts of gene III (red) or gene III-neg (blue) expressedfrom the respective promoters for a given amount of polymerase activity;therefore, fewer pIII molecules are generated from P_(T3) and morepIII-neg molecules are generated from P_(T7) for a given amount ofpolymerase activity as the experiment progresses. At t=0, host cellswith the DP and a P_(T3)-gene III AP are fed into the lagoon. For thefirst 12 hours, 200 ng/mL ATc was added to the lagoon to reduceselection stringency to zero, thereby enabling drift. At t=12 h, theconcentration of ATc was reduced to 20 ng/mL, resulting in an increasein selection stringency that allows weakly active variants to propagate.At t=28 h, host cells harboring an MP, a P_(T3)-gene III AP and ariboswitch-controlled P_(T7)-gene III-neg APneg were fed into thelagoons, initiating selection for variants capable of high levels ofactivity on P_(T3). In the absence of theophylline, transcriptionthrough the theophylline riboswitch-P_(T7) results in low levels ofpIII-neg production and therefore low negative selection pressureagainst P_(T7) recognition. At t=32, high levels of added theophylline(1 mM) was added to increase negative selection stringency, inducing arapid reduction in luciferase signal consistent with the loss ofpromiscuous RNAP variants. At t=52 host cells containing an MP, aP_(T3)-gene III AP with a weaker ribosome binding site (RBS), and aP_(T7)-gene III-neg APneg with enhanced RBS/higher origin of replicationcopy number were fed into the lagoons to further increase negative andpositive selection stringency. The in-line luminescence monitor was usedthroughout to infer population fitness (top). The luciferase signalagain dropped, consistent with the loss of intermediate specificityvariants from the pool, followed by rebound consistent with enrichmentof highly P_(T3)-specific RNAP variants.

FIG. 9. Activities of continuously evolved RNAP variants with alteredsubstrate specificities. Gene expression activities of randomly chosenRNAP clones (numbered along the X-axis in stages) isolated at the end ofthe drift stage (left, t=28 h), the low-stringency negative selectionwithout theophylline (center left, t=32 h), the low-stringency negativeselection with theophylline (center right, t=52 h), and thehigh-stringency negative selection (right, t=70.5 h) are shown. See FIG.10 for mutations present in each clone. N=no RNAP; T7=wild-type T7 RNAP.Gene expression activities on the T7 and T3 promoters of randomly chosenclones from each stage were measured (bottom). Gene expression data showmean values±s.e.m. for two replicates. A list of mutations present ineach clone is in FIG. 10. N, no RNAP; T7, wild-type T7 RNAP; NS,negative selection; mut, the arabinose-induced mutagenesis-enhancinggenes.

FIG. 10. Genotypes of continuously evolved RNAP variants. Mutationspresent in clones isolated following the drift stage (left, t=28 hrs),low-stringency negative selection without theophylline (center left,t=32 hrs), low-stringency negative selection with theophylline (centerright, t=52 hrs), and highstringency negative selection (right, t=70.5hrs). Mutations in blue are conserved in all clones and were found toconfer activity on P_(T3). Mutations in red are conserved in clones withhigh specificity for P_(T3) over P_(T7). Mutations in magenta are, as agroup, conserved and predicted based on the structure of T7 RNAP³⁰ to bephysically clustered, but mutually exclusive of each other (negativelyepistatic). Mutations in grey or black are isolated or modestlyconserved, respectively.

FIG. 11A-B. Analysis of evolved T7 RNAP mutations that confer P_(T3)specificity. (a) The gene expression activity in cells of T7 RNAPvariants containing subsets of mutations found in evolved clonesdescribed in FIG. 5 are shown on the T7 promoter (blue bars) and the T3promoter (red bars). (b) Location of evolved mutations.²¹ The T7promoter DNA is rendered as dark blue and light purple surfaces, withlight purple denoting nucleotide differences between the T7 and T3promoters. Cyan spheres identify evolved mutations that enable P_(T3)recognition. Red spheres identify mutations that evolved during negativeselection that contribute to specific recognition of P_(T3) over P_(T7).Magenta spheres represent a conserved cluster of mutually exclusivemutations evolved in clones following negative selection. The sequencesof the T7 and T3 promoters are shown at the bottom, with the differencesin red.

FIG. 12. Development of a DNA-binding continuous evolution system.Evaluation of a reporter system used to couple DNA binding to inductionof gene III-luciferase expression and a reporter system used to coupleDNA binding to an off-target sequence to production of pIIIneg-YFP.

FIG. 13. Optimization of a one-hybrid architecture for PACE. Comparisonof pIII-luciferase fold induction (ATc-induced Zif268expression/non-induced luminescence) resulting from binding of a Zif268fusion with either the α or ω subunit of RNAP to a Zif268 operatorsequence (5′-GCGTGGGCG-3′) centered at either −55 or −62. M refers to amedium-length linker between Zif268 and the RNAP subunit (AAATSGGGGAA,SEQ ID NO: XX), and L refers to a longer linker (AAGGGGSGGGGSGGGGSTAAA,SEQ ID NO: XX). Data represent mean+s.d. (n=3).

FIG. 14. Chromosomal pspBC deletion enables small-molecule control ofthe phage shock promoter response. Left panel: Comparison of phage-shockpromoter response between S1030 and S1632 cells. Upon phage infection,activation of a phage shock promoter (PSP) induces bacterial luciferaseexpression, and can be measured as an increase in luminescence. Thephage shock response sensors pspBC were deleted from S1632 cells,resulting in no transcriptional activation in the absence or presence ofinfecting phage. Right panel: Over-expression of pspBC from anarabinose-controlled promoter (PBAD) results in activation of the PSP ina manner independent of phage infection, eliminating variability intranscriptional activation of the promoter. Data represent mean±s.d.(n=3).

FIG. 15A-B. Generation of mutant PSP variants with altered dynamicrange. Mutants abrogating the efficiency or background transcription ofthe PSP were constructed and tested through low-level expression of thephage shock sensors pspBC, which are master inducers of the phage shockresponse. Generally, mutations were focused on the σ 54 core promoter.The “AR” series carried additional mutations to reduce the strength of σ70 cryptic promoters that may influence background transcription levels.(a) Luminescence signal in the presence or absence of 20 μM arabinosefrom wild-type and mutant PSP promoters. All readings were normalized towild-type PSP, which was set to 1. Data represent mean±s.d. (n=3). (b)Summary of activity, background levels, and genotypes of mutantpromoters assayed in (a). Background levels of all mutant promoters arelisted relative to wild-type.

FIG. 16A-C. Generation of 52060, a bacterial strain for chaperoneoverexpression and robust visualization of phage plaques. (a)Luminescence resulting from induction of a bacterial luciferase (luxAB)cassette driven by the P_(lux) promoter in response to the indicateddoses of N-(3-oxohexanoyl)-1-homoserine lactone (OHHL) (the LuxRtranscriptional regulator is also controlled by the P_(lux) promoter,only in the opposite direction). Data represent mean±s.d. (n=3). (b)Kinetic analysis of OHHL-mediated expression of GroESL (cassette:luxR-P_(lux)-groESL) on the folding of LuxAB (cassette:araC-P_(BAD)-LuxAB), a known substrate for GroESL. Increased in vivoconcentrations of GroESL result in improved folding of LuxAB and rapidsaturation of the luminescence response. (c) Comparison of the abilityto visualize plaque formation using S1030, S2058, S2059, and S2060cells. Chromosomally identical strains lacking (S1030) or carrying thelacZ and groESL cassettes (S2058, S2059, S2060) were infected with WTM13 bacteriophage. The modified strains carry the wild-type (WT) PSP,PSP-T1 or PSP-AR2, respectively. The reduced background and maintainedtranscriptional activation of the T1 and AR2 variants enables thevisualization of phage plaques in top agar supplemented with Bluo-Gal,an X-Gal derivative.

FIG. 17A-D. Continuous propagation of Zif268 in PACE, and reversion ofan inactive Zif268 mutant to wild-type. (a) Plaque assays of Zif268-SPor a control SP encoding T7 RNAP instead of Zif268 on S2060 cellscontaining APs encoding either the on- or off-target sequence, or S2208cells (positive control; see Example 2 for genotype information). (b)Schematic of the relative location of genes in the Zif268-SP, and asummary of mutations arising following 24 h of PACE to optimize thephage backbone and one-hybrid system. (c) Plaque assay results forwild-type Zif268-SP, inactive mutant Zif268-R24V-SP, and evolved SPsderived from a 24 h drift/24 h PACE experiment in the presence ofmutagenesis. ‘+’ denotes the presence of plaques, while ‘−’ denotes theabsence of plaques. (d) Genotypes of five phage clones isolatedfollowing PACE, all displaying reversion of V24 to R.

DEFINITIONS

The term “agent,” as used herein, refers to any molecule, entity, ormoiety. For example, an agent may be a protein, an amino acid, apeptide, a polynucleotide, a carbohydrate, a lipid, a detectable label,a binding agent, a tag, a metal atom, a contrast agent, a catalyst, anon-polypeptide polymer, a synthetic polymer, a recognition element, alinker, or chemical compound, such as a small molecule. In someembodiments, the agent is a binding agent, for example, a ligand, aligand-binding molecule, an antibody, or an antibody fragment.Additional agents suitable for use in embodiments of the presentinvention will be apparent to the skilled artisan. The invention is notlimited in this respect.

The term “detectable label” refers to a moiety that has at least oneelement, isotope, or functional group incorporated into the moiety whichenables detection of the molecule, e.g., a protein or peptide, or otherentity, to which the label is attached. Labels can be directly attachedor can be attached via a linker. It will be appreciated that the labelmay be attached to or incorporated into a molecule, for example, aprotein, polypeptide, or other entity, at any position. In general, adetectable label can fall into any one (or more) of five classes: I) alabel which contains isotopic moieties, which may be radioactive orheavy isotopes, including, but not limited to, ²H, ³H, ¹³C, ¹⁴C, ¹⁵N,¹⁸F, ³¹P, ³²P, ³⁵S, ⁶⁷Ga, ⁷⁶Br, ⁹⁹mTc (Tc-⁹⁹m), ¹¹¹In, ¹²³I, ¹²⁵I, ¹³¹I,¹⁵³Gd, ¹⁶⁹Yb, and ¹⁸⁶Re; II) a label which contains an immune moiety,which may be antibodies or antigens, which may be bound to enzymes(e.g., such as horseradish peroxidase); III) a label which is a colored,luminescent, phosphorescent, or fluorescent moieties (e.g., such as thefluorescent label fluorescein-isothiocyanate (FITC); IV) a label whichhas one or more photo affinity moieties; and V) a label which is aligand for one or more known binding partners (e.g.,biotin-streptavidin, FK506-FKBP). In certain embodiments, a labelcomprises a radioactive isotope, preferably an isotope which emitsdetectable particles, such as β particles. In certain embodiments, thelabel comprises a fluorescent moiety. In certain embodiments, the labelis the fluorescent label fluorescein-isothiocyanate (FITC). In certainembodiments, the label comprises a ligand moiety with one or more knownbinding partners. In certain embodiments, the label comprises biotin. Insome embodiments, a label is a fluorescent polypeptide (e.g., GFP or aderivative thereof such as enhanced GFP (EGFP)) or a luciferase (e.g., afirefly, Renilla, or Gaussia luciferase). It will be appreciated that,in certain embodiments, a label may react with a suitable substrate(e.g., a luciferin) to generate a detectable signal. Non-limitingexamples of fluorescent proteins include GFP and derivatives thereof,proteins comprising fluorophores that emit light of different colorssuch as red, yellow, and cyan fluorescent proteins. Exemplaryfluorescent proteins include, e.g., Sirius, Azurite, EBFP2, TagBFP,mTurquoise, ECFP, Cerulean, TagCFP, mTFP1, mUkG1, mAG1, AcGFP1, TagGFP2,EGFP, mWasabi, EmGFP, TagYPF, EYFP, Topaz, SYFP2, Venus, Citrine, mKO,mKO2, mOrange, mOrange2, TagRFP, TagRFP-T, mStrawberry, mRuby, mCherry,mRaspberry, mKate2, mPlum, mNeptune, T-Sapphire, mAmetrine, mKeima. See,e.g., Chalfie, M. and Kain, S R (eds.) Green fluorescent protein:properties, applications, and protocols Methods of biochemical analysis,v. 47 Wiley-Interscience, Hoboken, N.J., 2006; and Chudakov, D M, etal., Physiol Rev. 90(3):1103-63, 2010, for discussion of GFP andnumerous other fluorescent or luminescent proteins. In some embodiments,a label comprises a dark quencher, e.g., a substance that absorbsexcitation energy from a fluorophore and dissipates the energy as heat.

The term “mutation,” as used herein, refers to a substitution of aresidue within a sequence, e.g., a nucleic acid or amino acid sequence,with another residue, or a deletion or insertion of one or more residueswithin a sequence. Mutations are typically described herein byidentifying the original residue followed by the position of the residuewithin the sequence and by the identity of the newly substitutedresidue.

Continuous Evolution Concept

The term “continuous evolution,” as used herein, refers to an evolutionprocedure, in which a population of nucleic acids is subjected tomultiple rounds of (a) replication, (b) mutation, and (c) selection toproduce a desired evolved product, for example, a nucleic acid encodinga protein with a desired activity, wherein the multiple rounds can beperformed without investigator interaction and wherein the processesunder (a)-(c) can be carried out simultaneously. Typically, theevolution procedure is carried out in vitro, for example, using cells inculture as host cells. In general, a continuous evolution processprovided herein relies on a system in which a gene of interest isprovided in a nucleic acid vector that undergoes a life-cycle includingreplication in a host cell and transfer to another host cell, wherein acritical component of the life-cycle is deactivated and reactivation ofthe component is dependent upon a desired mutation in the gene ofinterest. Continuous evolution has been previously described inInternational Patent Application No. PCT/US2009/056194 andPCT/US2011/066747 and elsewhere^(1,6,7), each of which is herebyincorporated by reference in its entirety. An example of a continuousevolution experiment is PACE, as further summarized herein

The term “flow”, as used herein in the context of host cells, refers toa stream of host cells, wherein fresh host cells are being introducedinto a host cell population, for example, a host cell population in alagoon, remain within the population for a limited time and are thenremoved from the host cell population. In a simple form, a host cellflow may be a flow through a tube, or a channel, for example, at acontrolled rate. In other embodiments, a flow of host cells is directedthrough a lagoon that holds a volume of cell culture media and comprisesan inflow and an outflow. The introduction of fresh host cells may becontinuous or intermittent and removal may be passive, e.g., byoverflow, or active, e.g., by active siphoning or pumping. Removalfurther may be random, for example, if a stirred suspension culture ofhost cells is provided, removed liquid culture media will containfreshly introduced host cells as well as cells that have been a memberof the host cell population within the lagoon for some time. Eventhough, in theory, a cell could escape removal from the lagoonindefinitely, the average host cell will remain only for a limitedperiod of time within the lagoon, which is determined mainly by the flowrate of the culture media (and suspended cells) through the lagoon.

Since the viral vectors replicate in a flow of host cells, in whichfresh, uninfected host cells are provided while infected cells areremoved, multiple consecutive viral life cycles can occur withoutinvestigator interaction, which allows for the accumulation of multipleadvantageous mutations in a single evolution experiment.

The term “phage-assisted continuous evolution (PACE),” as used herein,refers to continuous evolution that employs selection phage as selectionviral vectors.

The term “high selection stringency” refers to conditions which favorrelatively active genetic variants and disfavor relatively weakly activeor inactive genetic variants. By performing the continuous evolutionconditions at a relatively high selection stringency, the bar for theactivity level required to pass the selection conditions is set at alevel that can exclude genetic variants that have low activity but highpotential to evolve greater activity in subsequent rounds of continuousevolution. Alternatively, relatively high selection stringencyencompasses the presence of a relatively high amount of the expressionproduct of a viral gene required to package a selection viral vectorinto an infectious viral particle (e.g., pIII protein) and its effect oninfectivity.

The term “low selection stringency” refers to conditions which favorrelatively weakly active or inactive genetic variants and disfavoractive genetic variants.

The term “gene variant thereof” refers to a variant that is at leastabout 50%, 60%, 70%, 80%, 90%, or 99% homologous to the original gene.

The term “dominant negative mutant” refers to a gene or gene variantthereof that encodes a gene product that antagonizes the gene product ofa phage gene that decreases or abolishes packaging of the selectionphagemids into infectious phage particles. Examples of a dominantnegative mutant is the N-C83 variant which is a pIII protein comprisingan N-C83 domain, which has an internal deletion of 70 amino acids (i.e.,amino acids 1-70) from the C-terminal domain of the pIII protein. Theamino acid sequence of the N-C83 variant is shown in FIG. 5, whichincludes other examples of pIII-neg variants. Other examples include thedominant negative mutant of the pIV protein (pIVneg) and other non-phageconventional counter selection genes. Alternative methods to a dominantnegative mutant include, for example, overexpression of phage proteinswhich, for example, bridge multiple interactions in phage packaging. Forexample, overexpression of pVI prevents pIII from being incorporated into the phage particle or overexpression of pIX/pVII may reduce theability of pVIII to get incorporated into the phage particle.

The term “negative selection,” as used herein, refers to the removal ofundesired mutations from the gene of interest.

The term “positive selection,” as used herein, refers to the retentionof desired mutations in the gene of interest.

The term “evolutionary drift,” as used herein, refers to theaccumulation of sequence differences that have minimal or no impact onthe fitness of an organism; such neutral mutations are random and arenot being actively selected or accumulation of mutations in the gene ofinterest that do not dramatically affect the activity or function ofsaid gene. For example, sequence polymorphisms arise randomly in apopulation, most of which have no effect on function. Stochasticprocesses allow a small fraction of these to increase in frequency untilthey are fixed in a population; these are detectable as neutralsubstitutions in interspecies comparisons. In some embodiments, a driftplasmid is used as described herein and the expression of a viral generequired to package the selection viral vector into infectious viralparticles is driven by a small molecule. Thus, evolutionary drift isallowed to occur by making the expression of a viral gene required topackage the selection viral vector into infectious viral particles to beindependent of the desired activity being evolved. A period of drift hasreduced (possibly to zero) selection stringency that allows evolvingsequences to “drift”, or mutate without regard to the selectionconsequences. Accumulation of mutations in the gene of interest in a waythat is independent of activity gene product of the gene of interest.

Viral Vectors

The term “viral vector,” as used herein, refers to a nucleic acidcomprising a viral genome that, when introduced into a suitable hostcell, can be replicated and packaged into infectious viral particlesable to transfer the viral genome into another host cell. The term viralvector extends to vectors comprising truncated or partial viral genomes.For example, in some embodiments, a viral vector is provided that lacksa gene encoding a protein essential for the generation of infectiousviral particles. In suitable host cells, for example, host cellscomprising the lacking gene under the control of a conditional promoter,however, such truncated viral vectors can replicate and generate viralparticles able to transfer the truncated viral genome into another hostcell. In some embodiments, the viral vector is a phage, for example, afilamentous phage (e.g., an M13 phage). In some embodiments, a viralvector, for example, a phage vector, is provided that comprises a geneof interest to be evolved.

The term “nucleic acid,” as used herein, refers to a polymer ofnucleotides. The polymer may include natural nucleosides (i.e,adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine,deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs(e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine,3-methyl adenosine, 5-methylcytidine, C5-bromouridine, C5-fluorouridine,C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine,C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine,8-oxoguanosine, O(6)-methylguanine, 4-acetylcytidine,5-(carboxyhydroxymethyl)uridine, dihydrouridine, methylpseudouridine,1-methyl adenosine, 1-methyl guanosine, N6-methyl adenosine, and2-thiocytidine), chemically modified bases, biologically modified bases(e.g., methylated bases), intercalated bases, modified sugars (e.g.,2′-fluororibose, ribose, 2′-deoxyribose, 2′-O-methylcytidine, arabinose,and hexose), or modified phosphate groups (e.g., phosphorothioates and5′-N-phosphoramidite linkages).

The term “protein,” as used herein refers to a polymer of amino acidresidues linked together by peptide bonds. The term, as used herein,refers to proteins, polypeptides, and peptides of any size, structure,or function. Typically, a protein will be at least three amino acidslong. A protein may refer to an individual protein or a collection ofproteins. Also, one or more of the amino acids in an inventive proteinmay be modified, for example, by the addition of a chemical entity suchas a carbohydrate group, a hydroxyl group, a phosphate group, a farnesylgroup, an isofarnesyl group, a fatty acid group, a linker forconjugation, functionalization, or other modification, etc. A proteinmay also be a single molecule or may be a multi-molecular complex. Aprotein may be just a fragment of a naturally occurring protein orpeptide. A protein may be naturally occurring, recombinant, orsynthetic, or any combination of these.

The term “gene to be evolved,” “gene of interest,” or “gene of interestto be evolved,” as used herein, are used interchangeably and refers to anucleic acid construct comprising a nucleotide sequence encoding a geneproduct of interest, for example, a gene product to be evolved in acontinuous evolution process as provided herein. In some embodiments,the gene product to be evolved has an evolved property such as activity,specificity, stability, or enantioselectivity. The term includes anyvariations of a gene of interest that are the result of a continuousevolution process according to methods provided herein. For example, insome embodiments, a gene of interest is a nucleic acid constructcomprising a nucleotide sequence encoding a protein to be evolved,cloned into a viral vector, for example, a phage genome, so that theexpression of the encoding sequence is under the control of one or morepromoters in the viral genome. In other embodiments, a gene of interestis a nucleic acid construct comprising a nucleotide sequence encoding aprotein to be evolved and a promoter operably linked to the encodingsequence. When cloned into a viral vector, for example, a phage genome,the expression of the encoding sequence of such genes of interest isunder the control of the heterologous promoter and, in some embodiments,may also be influenced by one or more promoters comprised in the viralgenome.

The term “function of a gene of interest,” as interchangeably used withthe term “property of a gene of interest,” refers to a property of agene product, for example, a nucleic acid or a protein, encoded by thegene of interest. For example, a function of a gene of interest may bean enzymatic activity (e.g., an enzymatic activity resulting in thegeneration of a reaction product, phosphorylation activity, phosphataseactivity, nuclease activity, methylase activity, glycosylation activity,etc.), altered and/or broadened substrate specificity, stability such asthermostability, enantioselectivity, an ability to activatetranscription (e.g., transcriptional activation activity targeted to aspecific promoter sequence), a bond-forming activity, (e.g., anenzymatic activity resulting in the formation of a covalent bond), or abinding activity (e.g., a protein, DNA, or RNA binding activity).

The terms “evolved product” and “evolution product” refer to a resultinggene product that was evolved from a gene of interest in a laboratoryevolution experiment. The resulting gene product can also be anintermediate gene product that will be subjected to further evolutionexperiments. A gene product can be an RNA or protein. For example, a T7RNAP that was evolved in a PACE experiment to be specific for the P_(T3)would be an evolution product.

The term “starting gene” or “starting genetic library,” as used herein,refers to a gene or library of one or more original starting gene(s)which are cloned into a viral genome (such as a phage genome) in theselection viral vector (such as a selection phage). Continuous evolutionstrategies are then used to evolve the starting genetic library into alibrary of evolved genes encoding evolution products. For example, theviral genome contains the gene of interest to be evolved, which may ormay not contain one or more mutations to begin with. A starting geneticlibrary can be a clonal population of single starting gene, or a libraryprepared by conventional methods, or a population from a previous roundof PACE. For example, the starting library can be generated by any meansof DNA manipulations, including but not limited to error-prone PCR,saturation mutagenesis, bacterial mutator strains or moderately modifiedlibraries based on prior knowledge of the biomolecule to be evolved.

The term “viral particle,” as used herein, refers to a viral genome, forexample, a DNA or RNA genome, that is associated with a coat of a viralprotein or proteins, and, in some cases, with an envelope of lipids. Insome embodiments, the viral particle is a phage particle. For example, aphage particle comprises a phage genome encapsulated by one or more coatproteins encoded by the wild-type phage genome.

The term “infectious viral particle,” as used herein, refers to a viralparticle able to transport its viral genome into a suitable host cell.Not all viral particles are able to transfer the viral genome to asuitable host cell. Particles unable to accomplish this are referred toas a non-infectious viral particles. In some embodiments, a viralparticle comprises a plurality of different coat proteins, wherein oneor some of the coat proteins can be omitted without compromising thestructure of the viral particle. In some embodiments, a viral particleis provided in which at least one coat protein cannot be omitted withoutthe loss of infectivity. If a viral particle lacks a protein thatconfers infectivity, the viral particle is not infectious. For example,an M13 phage particle that comprises a phage genome packaged in a coatof phage proteins (e.g., pVIII) but lacks pIII (protein III) is anon-infectious M13 phage particle because pIII is essential for theinfectious properties of M13 phage particles. In some embodiments, aninfectious viral particle is an infectious phage particle. In someembodiments, an infectious phage particle is an infectious M13 phageparticle.

The term “viral life cycle,” as used herein, refers to the viralreproduction cycle comprising insertion of the viral genome into a hostcell, replication of the viral genome in the host cell, and packaging ofa replication product of the viral genome into a viral particle by thehost cell. In some embodiments, a viral life cycle is a “phage lifecycle.”

In some embodiments, the viral vector provided is a phage. The terms“phage” and “bacteriophage” refer to a virus that infects bacterialcells. Typically, phages consist of an outer protein capsid enclosinggenetic material. The genetic material can be ssRNA, dsRNA, ssDNA, ordsDNA, in either linear or circular form. Phages and phage vectors arewell known to those of skill in the art, and non-limiting examples ofphages that are useful for carrying out the methods provided herein areλ (Lysogen), T2, T4, T7, T12, R17, M13, MS2, G4, P1, P2, P4, Phi X174,N4, Φ6, and Φ29. In certain embodiments, the phage utilized in thepresent invention is M13 phage. Additional suitable phages and hostcells will be apparent to those of skill in the art, and the inventionis not limited in this aspect. For an exemplary description ofadditional suitable phages and host cells, see Elizabeth Kutter andAlexander Sulakvelidze: Bacteriophages: Biology and Applications. CRCPress; 1^(st) edition (December 2004), ISBN: 0849313368; Martha R. J.Clokie and Andrew M. Kropinski: Bacteriophages: Methods and Protocols,Volume 1: Isolation, Characterization, and Interactions (Methods inMolecular Biology) Humana Press; 1^(st) edition (December, 2008), ISBN:1588296822; Martha R. J. Clokie and Andrew M. Kropinski: Bacteriophages:Methods and Protocols, Volume 2: Molecular and Applied Aspects (Methodsin Molecular Biology) Humana Press; 1^(st) edition (December 2008),ISBN: 1603275649; all of which are incorporated herein in their entiretyby reference for disclosure of suitable phages and host cells as well asmethods and protocols for isolation, culture, and manipulation of suchphages).

In some embodiments, the phage is a filamentous phage. In someembodiments, the phage is an M13 phage. M13 phages are well known tothose skilled in the art, and the biology of M13 phages has been studiedextensively. A schematic representation of the wild-type M13 genome isprovided in International Application No. PCT/US2011/066747, filed Dec.22, 2011, published as WO2012/088381. Wild-type M13 phage particlescomprise a circular, single-stranded genome of approximately 6.4 kb. Thewild-type genome includes eleven genes, gI-gXI, which, in turn, encodethe eleven M13 proteins, pI-pXI, respectively. gVIII encodes pVIII, alsooften referred to as the major structural protein of the phageparticles, while gIII encodes pIII, also referred to as the minor coatprotein, which is required for infectivity of M13 phage particles.

The M13 life cycle includes attachment of the phage to the conjugativepilus of a suitable bacterial host cell via the pIII protein andinsertion of the phage genome into the host cell. The circular,single-stranded phage genome is then converted to a circular,double-stranded DNA, also termed the replicative form (RF), from whichphage gene transcription is initiated. The wild-type M13 genomecomprises nine promoters and two transcriptional terminators as well asan origin of replication. This series of promoters provides a gradientof transcription such that the genes nearest the two transcriptionalterminators (gVIII and IV) are transcribed at the highest levels. Inwild-type M13 phage, transcription of all eleven genes proceeds in samedirection. One of the phage-encode proteins, pII, initiates thegeneration of linear, single-stranded phage genomes in the host cells,which are subsequently circularized, and bound and stabilized by pV. Thecircularized, single-stranded M13 genomes are then bound by pVIII at theinner bacterial membrane, while pV is stripped off the genome, whichinitiates the packaging process. At the end of the packaging process,multiple copies of pIII are attached to wild-type M13 particles, thusgenerating infectious phage ready to infect another host cell andconcluding the life cycle.

The M13 phage genome can be manipulated, for example, by deleting one ormore of the wild-type genes, and/or inserting a heterologous nucleicacid construct into the genome.

The M13 phage has been well characterized and the genomic sequence ofM13 has been reported. Representative M13 genomic sequences can beretrieved from public databases and an exemplary sequence is provided inentry V00604 of the National Center for Biotechnology Information (NCBI)database (www.ncbi.nlm.nih.gov). For example, an exemplary phage M13genome can be found under the GI number 56713234. The protein productfor gI, gII, gIII, gIV, gV, gVI, gVII, gVIII, gIX, gX, and gXI are alsoknown. The term “selection phage,” as used herein interchangeably withthe term “selection plasmid,” refers to a modified phage that comprisesa gene of interest to be evolved and lacks a full-length gene encoding aprotein required for the generation of infective phage particles. Forexample, some M13 selection phage provided herein comprise a nucleicacid sequence encoding a protein to be evolved, e.g., under the controlof an M13 promoter, and lack all or part of a phage gene encoding aprotein required for the generation of infective phage particles, e.g.,gI, gII, gIII, gIV, gV, gVI, gVII, gVIII, gIX, gX, or gXI, or anycombination thereof. For example, some M13 selection phage providedherein comprise a nucleic acid sequence encoding a protein to beevolved, e.g., under the control of an M13 promoter, and lack all orpart of a gene encoding a protein required for the generation ofinfective phage particles, e.g., the gIII gene encoding the pIIIprotein.

The term “helper phage,” as used herein interchangeable with the terms“helper phagemid” and “helper plasmid,” refers to an optional nucleicacid construct comprising a phage gene required for the phage lifecycle, or a plurality of such genes, but lacking a structural elementrequired for genome packaging into a phage particle. For example, ahelper phage may provide a wild-type phage genome lacking a phage originof replication. In some embodiments, a helper phage is provided thatcomprises a gene required for the generation of phage particles, butlacks a gene required for the generation of infectious particles, forexample, a full-length pIII gene. In some embodiments, the helper phageprovides only some, but not all, genes required for the generation ofphage particles. Helper phages are useful to allow modified phages thatlack a gene required for the generation of phage particles to completethe phage life cycle in a host cell. Typically, a helper phage willcomprise the genes required for the generation of phage particles thatare lacking in the phage genome, thus complementing the phage genome. Inthe continuous evolution context, the helper phage typically complementsthe selection phage, but both lack a phage gene required for theproduction of infectious phage particles.

The term “replication product,” as used herein, refers to a nucleic acidthat is the result of viral genome replication by a host cell. Thisincludes any viral genomes synthesized by the host cell from a viralgenome inserted into the host cell. The term includes non-mutated aswell as mutated replication products.

Accessory Plasmids, Drift Plasmids, and Helper Constructs

The term “accessory plasmid,” as used herein, refers to a plasmidcomprising a gene required for the generation of infectious viralparticles under the control of a conditional promoter. In one embodimentof the context of continuous evolution described herein, the conditionalpromoter of the accessory plasmid is typically activated by a functionof the gene of interest to be evolved. Accordingly, the accessoryplasmid serves the function of conveying a competitive advantage tothose viral vectors in a given population of viral vectors that carry agene of interest able to activate the conditional promoter. Only viralvectors carrying an “activating” gene of interest will be able to induceexpression of the gene required to generate infectious viral particlesin the host cell, and, thus, allow for packaging and propagation of theviral genome in the flow of host cells. Vectors carrying non-activatingversions of the gene of interest, on the other hand, will not induceexpression of the gene required to generate infectious viral vectors,and, thus, will not be packaged into infectious viral particles that caninfect host cells. In another embodiment, the conditional promoter isactivated by a small molecule inducer and its activation is independentof the gene of interest.

In some embodiments, the conditional promoter of the accessory plasmidis a activated by the transcriptional activity of which can be regulatedover a wide range, for example, over 2, 3, 4, 5, 6, 7, 8, 9, or 10orders of magnitude by the activating function, for example, function ofa protein encoded by the gene of interest. In some embodiments, thelevel of transcriptional activity of the conditional promoter dependsdirectly on the desired function of the gene of interest. This allowsfor starting a continuous evolution process with a viral vectorpopulation comprising versions of the gene of interest that only showminimal activation of the conditional promoter. In the process ofcontinuous evolution, any mutation in the gene of interest thatincreases activity of the conditional promoter directly translates intohigher expression levels of the gene required for the generation ofinfectious viral particles, and, thus, into a competitive advantage overother viral vectors carrying minimally active or loss-of-functionversions of the gene of interest.

The stringency of selective pressure imposed by the accessory plasmid ina continuous evolution procedure as provided herein can be modulated. Insome embodiments, the use of low copy number accessory plasmids resultsin an elevated stringency of selection for versions of the gene ofinterest that activate the conditional promoter on the accessoryplasmid, while the use of high copy number accessory plasmids results ina lower stringency of selection. The terms “high copy number plasmid”and “low copy number plasmid” are art-recognized, and those of skill inthe art will be able to ascertain whether a given plasmid is a high orlow copy number plasmid. In some embodiments, a low copy numberaccessory plasmid is a plasmid exhibiting an average copy number ofplasmid per host cell in a host cell population of about 5 to about 100.In some embodiments, a very low copy number accessory plasmid is aplasmid exhibiting an average copy number of plasmid per host cell in ahost cell population of about 1 to about 10. In some embodiments, a verylow copy number accessory plasmid is a single-copy per cell plasmid. Insome embodiments, a high copy number accessory plasmid is a plasmidexhibiting an average copy number of plasmid per host cell in a hostcell population of about 100 to about 5000. The copy number of anaccessory plasmid will depend to a large part on the origin ofreplication employed. Those of skill in the art will be able todetermine suitable origins of replication in order to achieve a desiredcopy number. The following table lists some non-limiting examples ofvectors of different copy numbers and with different origins ofreplication.

Plasmids pUC vectors pMB1* 500-700 high copy pBluescript ® vectors ColE1300-500 high copy pGEM ® vectors pMB1* 300-400 high copy pTZ vectorspMB1* >1000 high copy pBR322 and derivatives pMB1* 15-20 low copy pACYCand derivatives p15A 10-12 low copy pSC101 and derivatives pSC101 ~5very low copy *The pMB1 origin of replication is closely related to thatof ColE1 and falls in the same incompatibility group. The high-copyplasmids listed here contain mutated versions of this origin.

It should be understood that the function of the accessory plasmid,namely to provide a gene required for the generation of viral particlesunder the control of a conditional promoter the activity of whichdepends on a function of the gene of interest, can be conferred to ahost cell in alternative ways. Such alternatives include, but are notlimited to, permanent insertion of a gene construct comprising theconditional promoter and the respective gene into the genome of the hostcell, or introducing it into the host cell using an different vector,for example, a phagemid, a cosmid, a phage, a virus, or an artificialchromosome. Additional ways to confer accessory plasmid function to hostcells will be evident to those of skill in the art, and the invention isnot limited in this respect.

The term “drift plasmid,” as used herein refers to an accessory plasmidthat allows evolutionary drift to occur in response to a concentrationof a small molecule inducer. In some embodiments, the drift plasmidcontains a mutagenesis cassette under the control of another smallmolecule. In an embodiment, the drift plasmid contains a gene requiredto package the selection viral vector into an infectious viral particlesuch as gene III or a gene variant thereof.

The term “promoter” is art-recognized and refers to a nucleic acidmolecule with a sequence recognized by the cellular transcriptionmachinery and able to initiate transcription of a downstream gene. Apromoter can be constitutively active, meaning that the promoter isalways active in a given cellular context, or conditionally active,meaning that the promoter is only active in the presence of a specificcondition. For example, a conditional promoter may only be active in thepresence of a specific protein that connects a protein associated with aregulatory element in the promoter to the basic transcriptionalmachinery, or only in the absence of an inhibitory molecule. A subclassof conditionally active promoters are inducible promoters that requirethe presence of a small molecule “inducer” for activity. Examples ofinducible promoters include, but are not limited to, lactose-induciblepromoters (e.g., P_(lac)), arabinose-inducible promoters (P_(bad)),homoserine lactone-inducible promoters (e.g., P_(lux)),tetracycline-inducible promoters, and tamoxifen-inducible promoters. Avariety of constitutive, conditional, and inducible promoters are wellknown to the skilled artisan, and the skilled artisan will be able toascertain a variety of such promoters useful in carrying out the instantinvention, which is not limited in this respect.

The term “riboswitch” refers to regions of mRNA that adopt well definedsecondary and tertiary structures. They directly bind certain smallmolecules with high affinity and selectivity. Their cellular function isto repress or activate essential genes (for example those involved inbiosynthesis and transport of metabolites) in response to theintracellular level of their ligand. Ligand binding stabilizes either an“on” or “off” conformation which alters mRNA transcription, proteintranslation or mRNA splicing. Riboswitch can control gene expression atthe transcriptional or translational stage. In some embodiments, theexpression of a gene described herein, such as gene III, gene III-neg,is controlled through small molecule-RNA interactions. For example, geneexpression is controlled by a regulatable gene expression constructcomprising a nucleic acid molecule encoding an RNA comprising ariboswitch operably linked to a gene described herein, wherein theriboswitch comprises an aptamer domain and an expression platformdomain, wherein the riboswitch regulates expression of the genedescribed herein. For example, riboswitches are composed of an RNAaptamer domain which acts as a selective receptor for the binding of aspecific metabolite or small-molecule ligand. The binding of the ligandto aptamer induces a conformational change of the riboswitch that isable to adopt one of two possible conformations in response to ligandbinding leading to either an increase or decrease in the expression ofthe RNA transcript. Riboswitches can be naturally-occurring orsynthetic. Riboswitch can be responsive to various small molecules ormetabolites. For example, binding of hypoxanthine, guanine, or xanthineto a particular riboswitch can control transcription of genes. The smallmolecule can be endogenous or non-endogenous. For example,theophylline-activated riboswitches are useful for controlling thetranslation of a dominant negative mutant gene as described furtherherein.

Mutagens and Mutagenesis-Promoting Expression Constructs

The term “mutagen,” as used herein, refers to an agent that inducesmutations or increases the rate of mutation in a given biologicalsystem, for example, a host cell, to a level above thenaturally-occurring level of mutation in that system. Some exemplarymutagens useful for continuous evolution procedures are providedelsewhere herein and other useful mutagens will be evident to those ofskill in the art. Useful mutagens include, but are not limited to,ionizing radiation, ultraviolet radiation, base analogs, deaminatingagents (e.g., nitrous acid), intercalating agents (e.g., ethidiumbromide), alkylating agents (e.g., ethylnitrosourea), transposons,bromine, azide salts, psoralen, benzene,3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) (CAS no.77439-76-0), O,O-dimethyl-S-(phthalimidomethyl)phosphorodithioate(phos-met) (CAS no. 732-11-6), formaldehyde (CAS no. 50-00-0),2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) (CAS no. 3688-53-7),glyoxal (CAS no. 107-22-2), 6-mercaptopurine (CAS no. 50-44-2),N-(trichloromethylthio)-4-cyclohexane-1,2-dicarboximide (captan) (CASno. 133-06-2), 2-aminopurine (CAS no. 452-06-2), methyl methanesulfonate (MMS) (CAS No. 66-27-3), 4-nitroquinoline 1-oxide (4-NQO) (CASNo. 56-57-5), N4-Aminocytidine (CAS no. 57294-74-3), sodium azide (CASno. 26628-22-8), N-ethyl-N-nitrosourea (ENU) (CAS no. 759-73-9),N-methyl-N-nitrosourea (MNU) (CAS no. 820-60-0), 5-azacytidine (CAS no.320-67-2), cumene hydroperoxide (CHP) (CAS no. 80-15-9), ethylmethanesulfonate (EMS) (CAS no. 62-50-0),N-ethyl-N-nitro-N-nitrosoguanidine (ENNG) (CAS no. 4245-77-6),N-methyl-N-nitro-N-nitrosoguanidine (MNNG) (CAS no. 70-25-7),5-diazouracil (CAS no. 2435-76-9) and t-butyl hydroperoxide (BHP) (CASno. 75-91-2). Additional mutagens can be used in continuous evolutionprocedures as provided herein, and the invention is not limited in thisrespect.

Ideally, a mutagen is used at a concentration or level of exposure thatinduces a desired mutation rate in a given host cell or viral vectorpopulation, but is not significantly toxic to the host cells used withinthe average time frame a host cell is exposed to the mutagen or the timea host cell is present in the host cell flow before being replaced by afresh host cell.

The term “mutagenesis plasmid,” as used herein, refers to a plasmidcomprising a gene encoding a gene product that acts as a mutagen. Insome embodiments, the gene encodes a DNA polymerase lacking aproofreading capability. In some embodiments, the gene is a geneinvolved in the bacterial SOS stress response, for example, a UmuC,UmuD′, or RecA gene.

Host Cells

The term “host cell,” as used herein, refers to a cell that can host aviral vector useful for a continuous evolution process as providedherein. A cell can host a viral vector if it supports expression ofgenes of viral vector, replication of the viral genome, and/or thegeneration of viral particles. One criterion to determine whether a cellis a suitable host cell for a given viral vector is to determine whetherthe cell can support the viral life cycle of a wild-type viral genomethat the viral vector is derived from. For example, if the viral vectoris a modified M13 phage genome, as provided in some embodimentsdescribed herein, then a suitable host cell would be any cell that cansupport the wild-type M13 phage life cycle. Suitable host cells forviral vectors useful in continuous evolution processes are well known tothose of skill in the art, and the invention is not limited in thisrespect.

In some embodiments, modified viral vectors are used in continuousevolution processes as provided herein. In some embodiments, suchmodified viral vectors lack a gene required for the generation ofinfectious viral particles. In some such embodiments, a suitable hostcell is a cell comprising the gene required for the generation ofinfectious viral particles, for example, under the control of aconstitutive or a conditional promoter (e.g., in the form of anaccessory plasmid, as described herein). In some embodiments, the viralvector used lacks a plurality of viral genes. In some such embodiments,a suitable host cell is a cell that comprises a helper constructproviding the viral genes required for the generation of viralparticles. A cell is not required to actually support the life cycle ofa viral vector used in the methods provided herein. For example, a cellcomprising a gene required for the generation of infectious viralparticles under the control of a conditional promoter may not supportthe life cycle of a viral vector that does not comprise a gene ofinterest able to activate the promoter, but it is still a suitable hostcell for such a viral vector. In some embodiments, the viral vector is aphage and the host cell is a bacterial cell. In some embodiments, thehost cell is an E. coli cell. Suitable E. coli host strains will beapparent to those of skill in the art, and include, but are not limitedto, New England Biolabs (NEB) Turbo, Top10F′, DH12S, ER2738, ER2267, andXL1-Blue MRF′. These strain names are recognized in the art, and thegenotype of these strains has been well characterized. It should beunderstood that the above strains are exemplary only and that theinvention is not limited in this respect.

The term “fresh,” as used herein interchangeably with the terms“non-infected” or “uninfected” in the context of host cells, refers to ahost cell that has not been infected by a viral vector comprising a geneof interest as used in a continuous evolution process provided herein. Afresh host cell can, however, have been infected by a viral vectorunrelated to the vector to be evolved or by a vector of the same or asimilar type but not carrying the gene of interest.

In some embodiments, the host cell is a prokaryotic cell, for example, abacterial cell. In some embodiments, the host cell is an E. coli cell.In some embodiments, the host cell is a eukaryotic cell, for example, ayeast cell, an insect cell, or a mammalian cell. The type of host cell,will, of course, depend on the viral vector employed, and suitable hostcell/viral vector combinations will be readily apparent to those ofskill in the art.

In some PACE embodiments, for example, in embodiments employing an M13selection phage, the host cells are E. coli cells expressing theFertility factor, also commonly referred to as the F factor, sex factor,or F-plasmid. The F-factor is a bacterial DNA sequence that allows abacterium to produce a sex pilus necessary for conjugation and isessential for the infection of E. coli cells with certain phage, forexample, with M13 phage. For example, in some embodiments, the hostcells for M13-PACE are from the S109 strain with the genotype F′proA⁺B⁺Δ(lacIZY) zzf::Tn10(TetR)/endA1 recA1 galE15 galK16 nupG rpsL ΔlacIZYAaraD139 Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC) proBA::pir116λ⁻. Inanother example, the host cells are from the S1030 strain.

Lagoons, Cellstats, Turbidostats, Chemostats

The term “lagoon,” used herein interchangeably with the term “cellstat,” as used herein, refers to a vessel through which a flow of hostcells is directed. When used for a continuous evolution process asprovided herein, a lagoon typically holds a population of host cells anda population of viral vectors replicating within the host cellpopulation, wherein the lagoon comprises an outflow through which hostcells are removed from the lagoon and an inflow through which fresh hostcells are introduced into the lagoon, thus replenishing the host cellpopulation. In some embodiments, the flow of cells through the lagoon isregulated to result in an essentially constant number of host cellswithin the lagoon. In some embodiments, the flow of cells through thelagoon is regulated to result in an essentially constant number of freshhost cells within the lagoon.

The term “turbidostat,” as used herein, refers to a culture vesselcomprising host cells in suspension culture, in which the turbidity ofthe culture medium is substantially essentially constant over time. Insome embodiments, the turbidity of a suspension culture, for example, ofbacterial cells, is a measure for the cell density in the culturemedium. In some embodiments, a turbidostat comprises an inflow of freshmedia and an outflow, and a controller that regulates the flow intoand/or out of the turbidostat based on the turbidity of the suspensionculture in the turbidostat.

The term “chemostat,” as used herein, refers to a host cell culturesystem maintained at constant nutrient flow rate as opposed to a hostcell culture system maintained at constant turbidity (turbidostats).Chemostats also do not require turbidity monitoring as turbidostatsrequire.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

Some aspects of this invention provide methods for the continuousevolution of a biomolecule, for example, of a gene of interest (or geneto be evolved) or a gene product. Some aspects of this invention provideexperimental configurations, systems, apparatuses, reagents, software,and materials for the continuous evolution methods described herein.Vectors, vector systems, and kits for continuous evolution as describedherein are also provided. The continuous directed evolution methodsprovided herein utilize the following: 1) general modulation ofselection stringency to enable otherwise inaccessible properties ofbiomolecules to be evolved directly from weakly active/inactive startinggenes through a period of evolutionary drift; and/or 2) general negativeselection to enable counter-selection against undesired activities ofbiomolecules.

The foregoing new aspects related to selection stringency and negativeselection are useful because they expand the scope and capabilities ofcontinuous directed evolution strategies and address the needs ofpreviously described laboratory evolution experiments. For example, thenew aspects of continuous evolution methods described generally enablesthe evolution of enzymes with broadened and/or altered specificities.Prior strategies for continuous directed evolution, such as PACEdescribed below, have used relatively high selection stringency toevolve biomolecules with desired properties such as produce biomoleculeswith broadened specificity. Several needs were identified through theseearly efforts. First, it was observed that the use of high selectionstringency was not well suited for weakly active or inactive biomoleculevariants to be evolved. For example, previous PACE experiments evolvedT7 RNA polymerase (T7 RNAP) mutants that initiate transcription at theT3 promoter (P_(T3)), which differed from the T7 promoter (P_(T7)) atsix of 17 base positions.^(1,6,7) When wild-type T7 RNA polymerase waschallenged to evolve activity directly on P_(T3), active mutants did notemerge, and the phage in the lagoon washed out, indicating that thestringency of this initial selection was too high.^(1,6,7) Thesuccessful evolution of activity on P_(T3) instead required initialselection on a hybrid T7/T3 promoter (P_(T7/T3)) that served as anevolutionary stepping-stone toward the P_(T3) selection. For manypotential applications of PACE such as protein-protein interactions andbiosynthesis applications, suitable intermediate substrates may not beobvious or accessible. In addition, the methods herein may be useful forsubstrates in which the subcomponents are not simple, modular buildingblocks (e.g., small molecule substrates). To address this need, ageneral modulator of selection stringency was developed in whichcontinuous directed evolution such as PACE is initiated under reducedselection stringency to allow weakly active or inactive variants toaccess favorable mutations that enable propagation under subsequenthigher-selection stringency conditions. In some embodiments, theselection stringency is modulated by a small molecule inducer.

Second, it was further observed that the T7 RNAPs evolved to recognizeP_(T3) retained most or all of their activity on P_(T7). In the absenceof explicit negative selection against undesired activity, the evolutionof novel substrate recognition often results in enzymes with broadened,rather than truly altered specificity.^(8,9) For many potential targetsof PACE, including proteases, binding proteins, and genome engineeringenzymes, evolved proteins will require exceptional substrate specificityto function in complex cellular environments containing many potentialoff-target substrates. To address this need, a general negativeselection for PACE in which selection viral vectors such as a selectionphage (SP) encoding variants with undesired activities, such asoff-target substrate recognition, incur a replicative penalty.

Accordingly, provided herein are solutions that address the foregoingneeds associated with continuous directed evolution methods. Describedherein are strategies, systems, methods, reagents, and kits to expandthe scope and capabilities of continuous directed evolution and addressthe needs of previously described laboratory evolution experiments. Onesolution is to allow evolutionary drift to take place using reducedselection stringency. Another solution is to negatively select away fromundesired properties.

As further described in the Examples, T7 RNAP mutants that havewild-type like activity on P_(T3) and remarkable specificity for P_(T3)over P_(T7) have been evolved in a single PACE experiment. This evolveddegree of specificity exceeds that of wild-type T7 RNAP for P_(T7) overP_(T3), as well as that of wild-type T3 RNAP for P_(T3) over P_(T7). Thecombination of stringency modulation and negative selection enabled theevolution of polymerases with ˜10,000-fold changes in specificity in atotal time of three days. Together, these developments bring to PACE twocapabilities recognized to be important in certain laboratory evolutionefforts. These capabilities also expand the scope of PACE to include theevolution of biomolecules that possess radically altered properties suchas altered and highly specific new activities. For example, biomoleculesto be evolved include, e.g., nucleic acid binding proteins, proteases,recombinases, protein-protein interactions, genome engineering enzymes,biosynthetic clusters, endoribonucleases, and polymerases.

Overview of PACE Components

Phage-assisted continuous evolution (PACE), which has been previouslydescribed.^(1,6,7) Briefly, host cells continuously dilute an evolvingpopulation of bacteriophages in a fixed volume vessel (a “lagoon”)wherein each bacteriophage encodes a library member of an evolving gene.In general, dilution of the lagoon occurs faster than host cell divisionbut slower than phage replication, ensuring that the phage accumulatemutations. Each phage, referred to as a selection phage (SP), carries aprotein-encoding gene to be evolved instead of a phage gene (e.g., geneIII) that is required for infection. The phage gene required forinfection has been moved from the phage genome to an accessory plasmid(AP) carried by the host cells. Gene III encodes the phage protein pIII,which is essential for infectious phage production. Phage encodingactive variants trigger host-cell expression of gene III in proportionto the desired property (or “property of interest”) such that only SPencoding active library members induce the expression of pIII and theproduction of infectious progeny phage. Infectious progeny areconsequently produced, but phage encoding less-active variants produceless infectious progeny that are diluted out of the lagoon. Amutagenesis cassette is also provided to allow the gene of interest tobe mutated and evolved. In an embodiment, mutagenesis plasmid (MP),which contains a gene expression cassette encoding amutagenesis-promoting gene product, enables small-molecule control overthe mutation rate during phage replication. In another embodiment, themutagenesis cassette is found on the drift plasmid, as further describedherein. The host cell is contacted with the inducer, for example, in thelagoon, which induces expression of the mutagenesis promoting gene inthe host cells. Infectious progeny phage can infect fresh host cellsflowing into the lagoon and thereby continue to undergo additionalcycles of selection, replication, and mutation. A mutated replicationproduct of the viral vector, encoding an evolved protein, is isolatedfrom the population of host cells.

In some embodiments, the bacteriophage is an M13 filamentousbacteriophage. The pIII expression construct contains a conditionalpromoter regulating the expression of the pIII gene (i.e, gene III). Insome embodiments, the activity of the conditional promoter depends on adesired function of a gene product encoded by the gene of interest. Insome embodiments, the conditional promoter's activity can also dependson the presence of an inducer. For example, the conditional promoter isany inducible promoter known in the art such as an arabinose-induciblepromoter, wherein the inducer is arabinose.

The desired or undesired property of a gene product can be, for example,a binding activity or an enzymatic activity (such as a polymeraseactivity, a recombinase activity, a phosphotransferase activity, akinase activity, a phosphatase activity, or a protease activity). Forexample, in certain embodiments, it is the activity of the wild-typegene product such as cleavage of a wild-type protease cleavage site,phosphorylation of a wild-type site, recognition of substrate recognizedby the wild-type enzyme, etc. It could also be activity of the geneproduct of interest to a site/substrate that is similar/homologous tothe wild-type site/substrate or a site/substrate that issimilar/homologous to the target site/substrate of the evolution itself.

While selection phages (SP) and helper phages (HP) are described in thecontext of the PACE embodiment, it should be noted herein that SP and HPcan also refer to the corresponding counterparts for the viral-assistedcontinuous evolution experiments. The counterpart to SP is viralvectors, and the counterpart to HP is a helper plasmid.

In some embodiments, a gene of interest (or gene to be evolved) istransferred from cell to cell in a manner dependent on the activity ofthe gene of interest. In some embodiments, the gene of interest istransferred in a manner independent on the activity of the gene ofinterest. In some embodiments, the transfer vector is a virus orinfectious virus, for example, a bacteriophage. In some embodiments, theviral vector is a phage vector that infects bacterial host cells. Insome embodiments, the transfer vector is a retroviral vector, forexample, a lentiviral vector or a vesicular stomatitis virus vector thatinfects human or mouse cells. In some embodiments, the transfer vectoris a conjugative plasmid transferred from a donor bacterial cell to arecipient bacterial cell.

In some embodiments, the nucleic acid vector comprising the gene ofinterest is a phage, a viral vector, or naked DNA (e.g., a mobilizationplasmid). In some embodiments, transfer of the gene of interest fromcell to cell is via infection, transfect ion, transduction, conjugation,or uptake of naked DNA, and efficiency of cell-to-cell transfer (e.g.,transfer rate) is dependent on an activity of a product encoded by thegene of interest. For example, in some embodiments, the nucleic acidvector is a phage harboring the gene of interest, and the efficiency ofphage transfer (via infection) is dependent on an activity of the geneof interest in that a protein required for the generation of phageparticles (e.g., pIII for M13 phage) is expressed in the host cells onlyin the presence of the desired activity of the gene of interest. Inanother example, the nucleic acid vector is a retroviral vector, forexample, a lentiviral or vesicular stomatitis virus vector harboring thegene of interest, and the efficiency of viral transfer from cell to cellis dependent on an activity of the gene of interest in that a proteinrequired for the generation of viral particles (e.g., an envelopeprotein, such as VSV-g) is expressed in the host cells only in thepresence of the desired activity of the gene of interest. In anotherexample, the nucleic acid vector is a DNA vector, for example, in theform of a mobilizable plasmid DNA, comprising the gene of interest, thatis transferred between bacterial host cells via conjugation, and theefficiency of conjugation-mediated transfer from cell to cell isdependent on an activity of the gene of interest in that a proteinrequired for conjugation-mediated transfer (e.g., traA or traQ) isexpressed in the host cells only in the presence of the desired activityof the gene of interest. Host cells contain F plasmid lacking one orboth of those genes.

For example, some embodiments provide a continuous evolution system, inwhich a population of viral vectors comprising a gene of interest to beevolved replicates in a flow of host cells, e.g., a flow through alagoon, wherein the viral vectors are deficient in a gene encoding aprotein that is essential for the generation of infectious viralparticles, and wherein that gene is comprised in the host cell under thecontrol of a conditional promoter that can be activated by a geneproduct encoded by the gene of interest, or a mutated version thereof.In some embodiments, the activity of the conditional promoter depends ona desired function of a gene product encoded by the gene of interest.Viral vectors, in which the gene of interest has not acquired a mutationconferring the desired function, will not activate the conditionalpromoter, or only achieve minimal activation, while any mutation in thegene of interest that confers the desired mutation will result inactivation of the conditional promoter. Since the conditional promotercontrols an essential protein for the viral life cycle, activation ofthis promoter directly corresponds to an advantage in viral spread andreplication for those vectors that have acquired an advantageousmutation.

Modulation of Selection Stringency

Provided herein is a method for modulating the selection stringency inviral-assisted continuous evolution experiments. In some embodiments,the selection stringency is modulated by regulating the expression of agene required for the generation of infectious viral particles (e.g.,infectious phages). Generally, the gene required for the generation ofinfectious viral particles is on an accessory plasmid (AP) or on a driftplasmid (DP). A drift plasmid allows evolutionary drift to take place toevolve weakly active or inactive gene variants. The expression of a generequired for the generation of infectious viral particles produces aprotein required for the generation of infectious viral particles. Insome embodiments, the gene required for the generation of infectiousviral particles is gene III, which expresses the protein pIII needed togenerate infections phage. In some embodiments, the modulation of theselection stringency is independent of the desired activity to beevolved. In some embodiments, regulation of the expression of the generequired for the generation of infectious viral particles is under thecontrol of a small molecule inducible promoter (i.e.,chemically-regulated promoters) and therefore, is dependent on theconcentration of a small molecule. Examples of small molecule induciblepromoters are known in the scientific literature (see, e.g., Yamamotoet. al., 2001, Neurobiology of Disease, 8: 923-932). Non-limitingexamples of small molecule inducible promoters include lux promoters(e.g. P_(lux) from vibrio fishceri induced byN-(3-oxohexanoyl)-L-homoserine lactone (OHHL)); alcohol-regulatedpromoters (e.g., alcohol dehydrogenase I promoter (alcA), lac promoter(e.g., P_(lac)), arabinose-inducible promoters (e.g., P_(ara)),tetracycline-inducible promoters (e.g., P_(tet)), steroid-induciblepromoters, and tamoxifen-inducible promoters. In some embodiments, thesmall molecule inducible promoter is a TetA promoter (P_(tet)). In someembodiments, the small molecule is tetracycline or tetracycline analogs.In some embodiments, the small molecule is anhydrotetracycline (ATc). Inone embodiment, the small molecule is doxycycline. In one embodiment,the host cell drift promoter is partly a tetracycline-inducible promoter(P_(tet)), which drives expression of the TetR repressor and TetA, theprotein that pumps tetracycline out of the cell. In the absence oftetracycline or its analogs, TetR binds to the TetR operator sites andprevents transcription. In the presence of tetracycline or its analogs,TetR binds to tetracycline or a tetracycline analog, which induces aconformational change, making it unable to interact with the operator,so that target gene expression can occur.

In some embodiments, the host cell becomes viral infection-resistantprior to encountering the viral particle, thereby preventing viralpropagation. For example, low levels of pIII, such as the levelsexpressed at the beginning of a PACE experiment, have been shown torender cells resistant to filamentous phage infection.¹⁰ Accordingly,for situations where low levels of the protein (e.g., pIII) required forthe generation of infectious viral particles renders host cellsresistant to viral infection, it may be desirable to make the expressionof the protein (e.g., pIII) required for the generation of infectiousviral particles to be dependent on the condition that there be a priorviral infection of the host cells. In some embodiments, an E. coli phageshock promoter (P_(psp)) is used to require prior viral infection.Transcription from P_(psp) is induced by infection with filamentousphage via a pIV-dependent signaling cascade¹¹ or by overexpression of aplasmid-encoded phage pIV gene.

To produce a system in which protein expression requires both thepresence of the small molecule and prior viral infection, providedherein is a drift promoter. The drift promoter is located on a driftplasmid in the host cell. In some embodiments, the drift promoter isproduced from a P_(psp) variant with a TetR operator installed at aposition to disrupt either PspF or E. coli RNA polymerase binding. Insome embodiments, the TetR operator is placed adjacent to the +1transcription initiation site to produce a host cell drift promotercalled P_(psp-tet), which is induced only with the combination of phageinfection and ATc. In some embodiments, the P_(psp-tet) is placedupstream of the gene encoding the pIII protein. In some embodiments,propagation of the viral vector (e.g., SP) proceeds withoutactivity-dependent gene III expression. In some embodiments, SPs canpropagate in a small-molecule-dependent, activity-independent mannerusing the host cell drft promoter. In some embodiments, the driftpromoter is produced from one that is activated upon pspF release afterphage infection. In some embodiments, the host cell drift promoter isproduced from another promoter such as ones upstream of pal or hyfR.

In some embodiments, provided is a method of tuning the selectionstringency in continuous directed evolution methods. For example, totune the selection stringency, a host cell can use the followingplasmids: an activity-dependent AP, such as a P_(T7)-gene III AP, inwhich gene III is controlled by an activity-dependent promoter; and adrift plasmid (DP) with a host cell drift promoter-gene III cassette,such as a P_(psp-tet)-gene III. In some embodiments, the AP additionallycontains a reporter gene such as a luciferase gene. In some embodiments,the selection stringency is inversely proportional to the concentrationof the small molecule used. In some embodiments, low selectionstringency conditions are used. For example, saturating amounts of asmall molecule inducer (e.g., ATc) allows the P_(psp-tet)-gene IIIcassette in the DP to provide sufficient pIII to maximize phagepropagation, regardless of the SP-encoded property (such as activity),thus enabling genetic drift of the SP (low stringency). In someembodiments, an intermediate selection stringency is used. For example,at intermediate concentrations of a small molecule inducer (e.g., ATc),SPs encoding active library members have a replicative advantage over anSP encoding a weakly active/inactive variant by inducing additional pIIIexpression from an activity-dependent manner. An intermediateconcentration is determined by sampling a number of concentrations ofthe small molecule by using a plasmid the carries the native P_(tet)promoter driving bacterial luciferase. Intermediate concentrations aretypically considered those around the inflection point of a sigmoidalgraph. In some embodiments, high selection stringency conditions areused. For example, a zero or low amount of a small molecule inducer(e.g., ATc) allows the selection stringency to be determined by theactivity-dependent AP with no assistance from the P_(psp-tet)-gene IIIcassette (high stringency).

In some embodiments, the evolution experiments uses a ratio of SPs withactive starting genetic libraries to SPs with weakly active/inactivestarting genetic libraries of about 1:1, 1:5, 1:10, 1:20, 1:40, 1:60,1:80, 1:100, 1:120, 1:60, or 1:200. In one embodiment, the ratio of SPswith active to weakly active/inactive starting libraries is 1:100. Insome embodiments, phage population is generally followed over time usinga detectable label or directly via standard techniques. For example, thephage population can be followed using a combination of restrictionendonuclease digests and/or real-time measurements of luminescencemonitoring of promoter transcriptional activity (e.g., P_(T7)transcriptional activity), as further described herein. Additionalmethods are PCR, plaque assays, analysis by gel electrophoresis, oranalytical digestion. In some embodiments, an accessory plasmid carryingthe gene III and a gene encoding a co-expressed reporter fluorescentprotein (such as the luciferase gene, GFP, or other fluorescent proteindescribed herein) under the control of a conditional promoter (such as aP_(T7) or P_(T3)) would produce luminescence from the translatedluciferase when there is promoter transcriptional activity.

Selection stringency modulation can be used at any point in thecontinuous evolution process. In some embodiments, selection stringencymodulation is used towards the end of the continuous evolution process.In some embodiments, selection stringency modulation is used towards thebeginning of the continuous evolution process. In some embodiments, theselection stringency modulation is combined with negative selection.

In an embodiment, provided is a method for modulating the selectionstringency during viral-assisted evolution of a gene product, the methodcomprising: (a) introducing host cells into a lagoon, wherein the hostcell comprises a low selection stringency plasmid and a high selectionstringency plasmid, wherein the low selection stringency plasmidcomprises a viral gene required to package the selection viral vectorinto an infectious viral particles, wherein at least one gene requiredto package the selection viral vector into an infectious viral particlesis expressed in response to the a concentration of a small molecule, andwherein the high selection stringency plasmid comprises a second copy ofthe viral gene required to package the selection viral vector into theinfectious viral particles, wherein at least one viral gene required topackage the selection viral vector into an infectious viral particles isexpressed in response to a desired activity property of a gene productencoded by the gene to be evolved or an evolution product thereof; (b)introducing a selection viral vector comprising a gene to be evolvedinto a flow of host cells through a lagoon, wherein the gene to beevolved produces an active gene product or a weakly active or inactivegene product, wherein the active gene product has an activity thatdrives the expression of the viral gene required to package theselection viral vector into infectious viral particles in the highselection stringency plasmid and wherein the weakly active or inactivegene product has a relatively lower activity than the activity of theactive gene product; and (c) mutating the gene to be evolved within theflow of host cells, wherein the host cells are introduced through thelagoon at a flow rate that is faster than the replication rate of thehost cells and slower than the replication rate of the virus therebypermitting replication of the selection viral vector in the lagoon. Inan embodiment, the host cells are fed from a chemostat into the lagoon.

In an embodiment, the method further comprising isolating the selectionviral vector comprising an evolved product from the flow of cells anddetermining one or more properties of the evolved product. In oneembodiment, the low selection stringency plasmid contains a driftpromoter that is activated by a concentration of a small moleculeinducer and/or prior viral infection. In one embodiment, the highselection stringency plasmid contains a promoter that is activated by adesired property of a gene product encoded by the gene to be evolved oran evolution product thereof. In yet another embodiment, the lowselection stringency plasmid comprises a mutagenesis cassette under thecontrol of a small-molecule inducible promoter. In another embodiment,the low selection stringency plasmid allows a high level of evolutionarydrift to occur when the drift promoter is activated in response to aconcentration of a small molecule inducer and/or prior viral infection.In one embodiment, the high selection stringency plasmid allows a lowlevel of evolutionary drift to occur when the promoter is activated inresponse to a desired activity property of a gene product encoded by thegene to be evolved or an evolution product thereof.

In one embodiment, the property of the gene to be evolved originatedfrom a weakly active or inactive starting gene. In one embodiment, theproperty of the gene to be evolved originated from an active startinggene. In an embodiment, the high selection stringency comprises a T7promoter. In another embodiment, the low selection stringency comprisesa drift promoter that is activated by a small-molecule inducer and/orprior viral infection. In one embodiment, the drift promoter is aPpsp-tet promoter.

In one embodiment, the method of modulating the selection stringencyfurther comprises the use of negative selection and/or positiveselection.

Negative Selection

Selecting solely for activity on a new target substrate withoutcounter-selection against activity on non-target substrates is likely toresult in enzymes with broadened, rather than altered, specificity.Negative selection strategies that exert evolutionary pressure againstundesired activities, including poor specificity, are useful to avoid orminimize undesired (off-target) activity, and allow the evolution ofenzymes with high specificity, by linking undesired activities to theinhibition of viral or phage propagation or the inhibition of viral orphage infection.

Provided herein is a negative selection strategy which inhibitsinfectious viral production (e.g., phage production) in a manner that istunable and proportional to the ratio of undesired (off-target) todesired (on-target) activity. The selection is not dependent on theabsolute level of the undesired activity. In some embodiments, undesiredactivity induces expression of a protein that antagonizes the proteinproduct of a viral gene required for the generation of infectious viralparticles induced in a positive selection. In one embodiment, undesiredactivity induces expression of a protein that antagonizes the wild-typepIII protein induced in a positive selection. In some embodiments,undesired activity induces expression of a protein such as the dominantnegative variant of the pIV protein, or another specific phage gene suchas the pII and pVI proteins. In some embodiments, undesired activityinduces expression of a protein that reduces the ability of a viralparticle (e.g., phage) to be infectious. For example, a protein thatreduces the ability of a viral particle to be infectious is a dominantnegative protein. In some embodiments, expression of a dominant negativeform of pIII inhibits infectious phage production by blocking therelease of phage from the host cell.

Negative selection strategy for PACE in which undesired activities ofevolved products are penalized have been described in InternationalApplication No. PCT/US2011/066747, filed Dec. 22, 2011, published asWO2012/088381, which is incorporated herein by reference. In someembodiments, this is achieved by causing the undesired activity tointerfere with pIII production. For example, expression of an antisenseRNA complementary to the gIII RBS and/or start codon is one way ofapplying negative selection, while expressing a protease (e.g., TEV) andengineering the protease recognition sites into pIII is another.Negative selection is useful, for example, if the desired evolvedproduct is an enzyme with high specificity, for example, a transcriptionfactor or protease with altered, but not broadened, specificity. In someembodiments, negative selection of an undesired activity is achieved bycausing the undesired activity to interfere with pIII production, thusinhibiting the propagation of phage genomes encoding gene products withan undesired activity. In some embodiments, expression of adominant-negative version of pIII or expression of an antisense RNAcomplementary to the gIII ribosome binding site (RBS) and/or gIII startcodon is linked to the presence of an undesired activity. In someembodiments, a nuclease or protease cleavage site, the recognition orcleavage of which is undesired, is inserted into a pIII transcriptsequence or a pIII amino acid sequence, respectively. In someembodiments, a transcriptional or translational repressor is used thatrepresses expression of a dominant negative variant of pIII andcomprises a protease cleavage site the recognition or cleavage of whichis undesired.

In some embodiments, counter-selection against activity on non-targetsubstrates is achieved by linking undesired evolved product activitiesto the inhibition of phage propagation. For example, in someembodiments, in which a transcription factor is evolved to recognize aspecific target sequence, but not an undesired off-target sequence, anegative selection cassette is employed, comprising a nucleic acidsequence encoding a dominant-negative version of pIII (pIII-neg) underthe control of a promoter comprising the off-target sequence. If anevolution product recognizes the off-target sequence, the resultingphage particles will incorporate pIII-neg, which results in inhibitionof phage infective potency and phage propagation, thus constituting aselective disadvantage for any phage genomes encoding an evolutionproduct exhibiting the undesired, off-target activity, as compared toevolved products not exhibiting such an activity. In some embodiments, adual selection strategy is applied during a continuous evolutionexperiment, in which both positive selection and negative selectionconstructs are present in the host cells. In some such embodiments, thepositive and negative selection constructs are situated on the sameplasmid, also referred to as a dual selection accessory plasmid. In somesuch embodiments, the positive and negative selection constructs aresituated on two different accessory plasmids within a host cell. The useof two separate accessory plasmids gives the ability to modulate thepositive and negative selection aspects independently, thereby yielding,for example, highly selective variants of the gene of interest.

For example, in some embodiments, a dual selection accessory plasmid isemployed comprising a positive selection cassette, comprising apIII-encoding sequence under the control of a promoter comprising atarget nucleic acid sequence, and a negative selection cassette,comprising a pIII-neg encoding cassette under the control of a promotercomprising an off-target nucleic acid sequence. In other embodiments, afirst accessory plasmid (i.e, a positive selection AP) is employedcomprising a positive selection cassette, comprising a pIII-encodingsequence under the control of a promoter comprising a target nucleicacid sequence, and a second accessory plasmid (i.e, a negative selectionAP) is employed comprising a negative selection cassette, comprising apIII-neg encoding cassette under the control of a promoter comprising anoff-target nucleic acid sequence. One advantage of using a simultaneousdual selection strategy is that the selection stringency can befine-tuned based on the activity or expression level of the negativeselection construct as compared to the positive selection construct.Another advantage of a dual selection strategy is the selection is notdependent on the presence or the absence of a desired or an undesiredactivity, but on the ratio of desired and undesired activities, and,thus, the resulting ratio of pIII and pIII-neg that is incorporated intothe respective phage particle.

Some aspects of this invention provide or utilize a dominant negativevariant of pIII (pIII-neg), previously described in InternationalApplication No. PCT/US2011/066747, filed Dec. 22, 2011, published asWO2012/088381, which is incorporated herein by reference. These aspectsare based on the discovery that a pIII variant (N-C83) that comprisesthe two N-terminal domains of pIII and a truncated,termination-incompetent C-terminal domain is not only inactive but is adominant-negative variant of pIII. The mutated C domain has an internaldeletion of 70 amino acids. A pIII variant comprising the two N-terminaldomains of pIII and a truncated, termination-incompetent C-terminaldomain was described in Bennett, N. J.; Rakonjac, J., Unlocking of thefilamentous bacteriophage virion during infection is mediated by the Cdomain of pIII. Journal of Molecular Biology 2006, 356 (2), 266-73; theentire contents of which are incorporated herein by reference. Withoutwishing to be bound by theory, some aspects of this invention are basedin part on the discovery that a pIII-neg variant as provided herein issufficient to mediate attachment to a phage particle but cannot catalyzethe detachment of nascent phage from the host cell membrane during phageparticle synthesis. Accordingly, such pIII-neg variants are useful fordevising a negative selection strategy in the context of PACE, forexample, by providing an expression construct comprising a nucleic acidsequence encoding a pIII-neg variant under the control of, for example,a promoter comprising a recognition motif, the recognition of which isundesired. In other embodiments, the undesired substrate or undirectedsubstrate recognition is tethered upstream of a dominant negative mutantsuch as pIII-neg.

In some embodiments, multiple undesired activities, e.g., off-targetDNA-binding activities, of a gene product to be evolved are selectedagainst using the negative selection strategies provided herein. Anegative selection against multiple off-target DNA-binding activitiescan be achieved in different ways. For example, a selection phage may bepropagated initially in host cells carrying an accessory plasmid linkingactivity towards a first undesired off-target site to negativeselection, and subsequently in host cells carrying different accessoryplasmids creating negative selective pressure against differentoff-target sites. In addition to such sequential approaches, thisdisclosure also provides strategies featuring a combination of differentnegative selection constructs to create negative selective pressureagainst multiple off-target activities. For example, in someembodiments, a selection phage is propagated during a PACE experiment inhost cells comprising two or more accessory plasmids, each onecontaining a negative selection construct linking DNA-binding to adifferent undesired off-target site to negative selection, thusresulting in selective pressure against two or more different off-targetsites. In some embodiments, a selection phage is propagated in apopulation of host cells in which different host cells carry differentaccessory plasmids, e.g., linking activity towards different undesiredoff-target sites to negative selection. While each host cell in such apopulation will only create negative selective pressure against a singleoff-target activity, the propagation in a population of host cellscarrying different accessory plasmids results in a selective pressureagainst multiple off-target activities.

In other embodiments, pIII-neg is used in a positive selection strategy,for example, by providing an expression construct in which a pIII-negencoding sequence is controlled by a promoter comprising a nucleasetarget site or a repressor recognition site, the recognition of eitherone of which is desired.

The methods herein can also use positive and negative selectionstrategies which can be used alternately with one another or usedsimultaneously. The positive and negative selection schemes would beidentical for a PACE application of interest, with the only differencebeing the positive selection results in the synthesis of pIII and thenegative selection results in the synthesis of pIII-neg. For example,the expression of one or the other is dependent upon the selectionscheme.

Positive and negative selection strategies can further be designed tolink non-DNA directed activities to phage propagation efficiency. Forexample, protease activity towards a desired target protease cleavagesite can be linked to pIII expression by devising a repressor of geneexpression that can be inactivated by a protease recognizing the targetsite. In some embodiments, pIII expression is driven by a promotercomprising a binding site for such a repressor. Suitable transcriptionalrepressors are known to those in the art, and one exemplary repressor isthe lambda repressor protein that efficiently represses the lambdapromoter pR and can be modified to include a desired protease cleavagesite (see, e.g., Sices, H. J.; Kristie, T. M., A genetic screen for theisolation and characterization of site-specific proteases. Proc NatlAcad Sci USA 1998, 95 (6), 2828-33; and Sices, H. J.; Leusink, M. D.;Pacheco, A.; Kristie, T. M., Rapid genetic selection ofinhibitor-resistant protease mutants: clinically relevant and novelmutants of the HIV protease. AIDS Res Hum Retroviruses 2001, 17 (13),1249-55, the entire contents of each of which are incorporated herein byreference). The lambda repressor (cI) contains an N-terminal DNA bindingdomain and a C-terminal dimerization domain. These two domains areconnected by a flexible linker. Efficient transcriptional repressionrequires the dimerization of cI, and, thus, cleavage of the linkerconnecting dimerization and binding domains results in abolishing therepressor activity of cI.

Some embodiments provide a pIII expression construct that comprises a pRpromoter (containing cI binding sites) driving expression of pIII. Whenexpressed together with a modified cI comprising a desired proteasecleavage site in the linker sequence connecting dimerization and bindingdomains, the cI molecules will repress pIII transcription in the absenceof the desired protease activity, and this repression will be abolishedin the presence of such activity, thus providing a linkage betweenprotease cleavage activity and an increase in pIII expression that isuseful for positive PACE protease selection. Some embodiments provide anegative selection strategy against undesired protease activity in PACEevolution products. In some embodiments, the negative selection isconferred by an expression cassette comprising a pIII-neg encodingnucleic acid under the control of a cI-repressed promoter. Whenco-expressed with a cI repressor protein comprising an undesiredprotease cleavage site, expression of pIII-neg will occur in a cellharboring phage expressing a protease exhibiting protease activitytowards the undesired target site, thus negatively selecting againstphage encoding such undesired evolved products. A dual selection forprotease target specificity can be achieved by co-expressingcI-repressible pIII and pIII-neg encoding expression constructs withorthogonal cI variants recognizing different DNA target sequences, andthus allowing for simultaneous expression without interfering with eachother. Orthogonal cI variants in both dimerization specificity andDNA-binding specificity are known to those of skill in the art (see,e.g., Wharton, R. P.; Ptashne, M., Changing the binding specificity of arepressor by redesigning an alphahelix. Nature 1985, 316 (6029), 601-5;and Wharton, R. P.; Ptashne, M., A new-specificity mutant of 434repressor that defines an amino acid-base pair contact. Nature 1987, 326(6116), 888-91, the entire contents of each of which are incorporatedherein by reference).

Provided herein is negative selection of a biomolecule with an undesiredactivity in combination with positive selection of a biomolecule withdesired activity, wherein enrichment of the biomolecule with a desiredactivity is controlled by, for example, the concentration of a smallmolecule. In some embodiments, a host cell contains both a positiveselection AP and a negative selection AP, wherein the positive selectionAP contains a viral gene required for the generation of infectious viralparticles (e.g., phage gene such as gene III) and wherein the negativeselection AP contains a dominant negative mutant of the viral gene(e.g., gene III-neg). In some embodiments, the viral gene required forthe generation of infectious viral particles contained in the positiveselection AP is under the control of a first small molecule induciblepromoter and the dominant negative mutant of the viral gene contained inthe negative selection AP is under the control of a second smallmolecule inducible promoter, wherein a first small molecule is used toinduce the first small molecule inducible promoter, and wherein a secondsmall molecule is used to induce the second small molecule induciblepromoter. In one embodiment, the first small molecule inducible promoteris a TetA promoter (P_(tet)). In one embodiment, the small molecule usedto induce transcription of genes under the control of P_(tet) is ATc. Inone embodiment, the second small molecule inducible promoter is apromoter of the lac operon (P_(lac)). In one embodiment, the smallmolecule used to induce transcription of genes under the P_(lac) isisopropyl β-D-1-thiogalactopyranoside (IPTG). In some embodiments, thenegative selection AP containing a dominant negative mutant of a phagegene is under the control of a riboswitch. Non-limiting examples ofriboswitches include, for example, the lysine riboswitch from Bacillussubtilis, the glycine riboswitch from Bacillus subtilis, the adenineriboswitch from Bacillus subtilis or the TPP tandem riboswitch fromBacillus anthracia. In addition to the foregoing naturally occurringriboswitch elements, synthetic riboswitch elements can also be used,such as, for example, the theophylline riboswitch, the biotin riboswitchor the Tet riboswitch. In some embodiments, the riboswitch is activatedby theophylline. In some embodiments, the rate of enrichment of thebiomolecule with desired activity is dependent on the concentration ofthe small molecule used to control translation of the dominant negativeprotein. In some embodiments, the phage gene is gene III. In someembodiments, the dominate negative mutant of a phage gene is a geneencoding a dominant negative pIII protein. In some embodiments, thedominant negative pIII protein contains a mutant C domain. In someembodiments, the dominant negative pIII protein is the N-C83 variant,wherein the C domain of the pIII protein has an internal deletion of acertain number of 70 amino acids.

In some embodiments, the viral gene required for the generation ofinfectious viral particles contained in the positive selection AP isunder the control of a promoter comprising a desired DNA binding sitefor the gene product. In some embodiments, following initial negativeselection, high-stringency host cells are used to further enhance thedesired activity, wherein the positive selection construct has beenmodified to reduce the translation of pIII from the AP and wherein thenegative selection construct has been modified to enhance thetranslation of pIII-neg from the AP_(neg). For example, the RBS on thepositive selection AP is weakened; a strong RBS replaces the riboswitchon the negative selection AP; or the negative selection AP has beenmodified to be a high copy plasmid.

In negative and positive selection methods herein, the wild-type pIIIand pIII-neg compete for incorporation into a progeny phage particle,thus making the potency of inhibition sensitive to the ratio of thedesired or undesired activities.

The ratio of the SPs containing desired and undesired activity can bevaried in the evolution experiments. In some embodiments, the ratio of aSP containing a gene encoding a biomolecule with desired activity to aSP containing a gene encoding a biomolecule with undesired activity isabout 1:1, 1:100, 1:10³, 1:10⁴, 1:10⁵, 1:10⁶, 1:10⁹ or 1:10¹⁰. In oneembodiment, the ratio of desired SP to undesired SP is about 1:10⁶.

In some embodiments, the number of hours of PACE with negative selectionto evolve a biomolecule with desired activity is about 3 hours, 4 hours,5 hours, 6 hours, 8 hours, 10 hours, 15 hours, 20 hours, 30 hours, or 40hours. In some embodiments, the number of hours of PACE with negativeselection to evolve a biomolecule with desired activity is about 4 toabout 8 hours.

Negative selection can be used at any point in the continuous evolutionprocess. In some embodiments, negative selection is used towards the endof the continuous evolution process. In some embodiments, negativeselection is used towards the beginning of the continuous evolutionprocess. In some embodiments, the negative selection is combined withselection stringency modulations.

In an embodiment, provided is a method of using negative selectionduring viral-assisted evolution of a gene product, the methodcomprising: (a) introducing host cells into a lagoon, wherein the hostcell comprises a negative selection gene and a dominant negative mutantgene of a phage gene that decreases or abolishes packaging of theselection viral vector into infectious viral particles, wherein thedominant negative gene is expressed in response to an undesired activityof the gene to be evolved or an evolution product thereof or in responseto a concentration of a small molecule inducer; (b) introducing amixture of selection viral vectors into a flow of host cells through alagoon, wherein one type of selection viral vector comprises a negativeselection gene comprising an undesired gene or gene encoding anundesired property of the gene to be evolved and wherein another type ofselection viral vector comprises a positive selection gene comprising adesired gene or gene encoding a desired property of the gene to beevolved; and (c) mutating the gene to be evolved within the flow of hostcells, wherein the host cells are introduced through the lagoon at aflow rate that is faster than the replication rate of the host cells andslower than the replication rate of the virus thereby permittingreplication of the selection viral vector in the lagoon. In oneembodiment, the negative selection gene and dominant negative mutantgene is found on an negative selection accessory plasmid. In anembodiment, the negative selection accessory plasmid further comprises asequence encoding for a riboswitch. In another embodiment, thetranscribed riboswitch is activated by a small-molecule. In anembodiment, the small-molecule is theophylline.

In one embodiment, the negative selection accessory plasmid comprises ahigh-copy origin of replication. In an embodiment, the negativeselection accessory plasmid comprises a modulation of theribosome-binding site driving translation of the dominant negativemutant gene, wherein modulation is weakening or strengthening. Inanother embodiment, the negative selection accessory plasmid furthercomprises a gene encoding for a detectable label. In an embodiment, thedetectable label is a fluorescent protein. In a specific embodiment, thefluorescent protein is luciferase.

In another embodiment, the dominant negative mutant gene is expressedunder the control of a theophylline-activated riboswitch. In anembodiment, the negative selection gene is a promoter. In oneembodiment, the negative selection promoter is a T3 or T7 promoter.

In one embodiment, the method of negative selection further comprises amethod of positive selection, the method comprising a positive selectiongene comprising a gene that decreases or abolishes packaging of theselection phagemids into infectious phage particles. In an embodiment,the positive selection gene is found on a positive selection accessoryplasmid. In one embodiment, the positive selection accessory plasmidcomprises modulation of the ribosome binding site, wherein modulation isweakening or strengthening. In another embodiment, the positiveselection accessory plasmid further comprises a sequence encoding for ariboswitch. In one embodiment, the transcribed riboswitch is activatedby a small-molecule. In an embodiment, the small-molecule istheophylline.

In one embodiment, the positive selection accessory plasmid furthercomprises a gene encoding for a detectable label. In another embodiment,the detectable label is a fluorescent protein. In one embodiment, thefluorescent protein is luciferase.

In an embodiment, the positive selection accessary plasmid and negativeselection plasmid are on the same or different plasmids. In anembodiment, the positive selection gene is a promoter. In anotherembodiment, the positive selection promoter is a T7 or T3 promoter.

In one embodiment, the host cells further comprises a mutagenesiscassette located on a mutagenesis plasmid and is under control of asmall-molecule inducible promoter. In one embodiment, the host cellsintroduced into the lagoon are from a chemostat.

In an embodiment, wherein the small molecule inducer that drivesexpression of a positive selection gene is different from the smallmolecule inducer that drives expression of a dominant negative mutantgene. In an embodiment, the selection viral vector comprising a negativeselection gene and the selection viral vector comprising a positiveselection gene is found in a ratio of about 1:1 to about 10⁹:1.

In one embodiment, the dominant negative mutant is found on an accessoryplasmid. In an embodiment, the positive selection gene is found on thesame or different accessory plasmid as the accessory plasmid comprisingthe dominant negative mutant. In one embodiment, the dominant negativemutant gene encodes a dominant negative mutant protein that antagonizesthe gene product of a phage gene that decreases or abolishes packagingof the selection phagemids into infectious phage particles. In oneembodiment, the dominant negative mutant gene is a dominant negativemutant of a gene encoding a dominant negative pIII protein. In oneembodiment, the dominant negative pIII protein comprises a mutantC-domain. In one embodiment, the dominant negative pIII proteincomprises a deletion of amino acids 1-70 from the C-domain. In anembodiment, the dominant negative pIII protein is the N-C83 variant. Inanother embodiment, the positive selection gene is a gene encoding apIII protein.

Methods and Vector Systems for Modulation of Selection Stringency andNegative Selection

Provided herein is a method of rapidly evolving biomolecules, such asenzymes, polymerases, and transcription factors, with alteredproperties, such as altered substrate specificity or activity, using thetunable selection stringency and negative selection methods describedherein. In some embodiments, selection stringency strategies are used togenerate an initial evolved product prior to using negative selectionmethods to generate enhanced evolved product. Provided herein are alsovector systems which combine the selection stringency modulation andnegative selection strategies described herein.

In one embodiment, provided is a method of modulating the selectionstringency and using negative selection and/or positive selection duringviral-assisted evolution of a gene product, the method comprising theforegoing methods described. In an embodiment, modulating the selectionstringency and using negative selection and/or positive are used invarious stages of a continuous evolution experiment.

In an embodiment, the gene of interest to be evolved encodes aDNA-binding gene product. In one embodiment, the DNA-binding geneproduct is an RNA polymerase. In a specific embodiment, the RNApolymerase is a T7 RNA polymerase. In an embodiment, the T7 RNApolymerase has been evolved to be specific for the T3 promoter. In anembodiment, expression of the gene required to package the selectionphagemid into infectious particles is driven by a promoter comprising adesired DNA binding site for the gene product. In one embodiment,expression of the dominant negative mutant of the phage gene thatdecreases or abolishes packaging of the selection phagemids intoinfectious phage particles is driven by a promoter comprising anundesired DNA binding site for the gene product. In an embodiment, thehost cells are introduced into lagoons from a chemostat. In oneembodiment, the host cells are prokaryotic cells amenable to phageinfection, replication, and production. In one embodiment, the host cellis bacterial. In another embodiment, the host cell is E. coli. Inanother embodiment, a gene that is required to package the selectionviral vector into infectious viral particles is a gene that encodes forthe pIII protein. In one embodiment, the gene that encodes for the pIIIprotein is gene III or a gene that is at least about 50%, 60%, 70%, 80%,90%, or 99% homologous to gene III. In another embodiment, the selectionviral vector comprises all viral genes required for the generation ofviral particles, except for a full-length gene that is required topackage the selection viral vector into infectious viral particles. Inone embodiment, the selection viral vector is a selection phage orphagemid. In another embodiment, the selection viral vector is afilamentous phage. In one embodiment, the selection viral vector is M13phage. In another embodiment, the undesired DNA binding site is PT7 orPT3. In an embodiment, the desired DNA binding site is PT7 or PT3.

In some embodiments, the use of selection stringency modulation methodsallows the continuous directed evolution of a first evolved productcontaining a novel property evolved from a biomolecule with weaklyactive/inactive starting activity. In some embodiments, the use ofintermediate substrates in the evolution is not needed. In oneembodiment, a low selection stringency is used. In one embodiment, afirst population of host cells carries a vector system containing acombination of a P_(T3)-gene III AP and a DP containing the P_(psp-tet)gene III cassette and the SP-T7_(WT) carries a biomolecule with inactivestarting activity (i.e., T7_(WT) RNAP) because it has negligiblestarting activity on P_(T3). In one embodiment, the AP contains areporter gene such as a luciferase gene. In one embodiment, the firstevolved product is a T7 RNAP that recognizes P_(T3). In someembodiments, an intermediate substrate such as a hybrid T7/T3 promoteris not needed in the evolution of toward P_(T3) selection.

In some embodiments, the subsequent use of negative selection methodsallows the evolution of a second evolved product containing propertiesthat is altered or enhanced from the initial properties found in thefirst evolved product. In one embodiment, a second population of hostcells carries the same P_(T3)-gene III AP, a MP without the driftcassette, and a P_(T7)-gene III-neg AP_(neg), wherein pIII-negexpression is driven by P_(T7) transcription and controlled by atheophylline-dependent riboswitch. In one embodiment, the AP contains areporter gene such as a luciferase gene. Negative selection is activatedby addition of theophylline to the lagoon. The result is a secondevolved product that demonstrates specificity improvements for P_(T3)and decreased specificity for P_(T7).

In some embodiments, the property of the second evolved product isfurther altered or enhanced by increasing the stringency of the negativeselection. In some embodiments, the increased stringency is enhanced byincreasing the ratio of a dominant negative mutant to the positivemutant counterpart. In some embodiments, the stringency of negativeselection can be controlled by varying the strength of the RBS and theAP copy number. In some embodiments, the stringency of negativeselection is increased using high-stringency host cells containing thesame MP without the drift cassette, a P_(T3)-gene III AP with a reducedRBS, and a P_(T7)-gene III-neg APneg containing a high-copy pUC originand a strong RBS. In one embodiment, the AP contains a reporter genesuch as a luciferase gene. The foregoing exemplary modificationsincrease the stringency of negative selection by increasing the ratio ofpIII-neg to pIII expressed in the host cell.

Evolved Product

Some aspects of this invention provide evolved products (or evolutionproducts) of continuous evolution processes described herein, whereinthe evolved products contain evolved properties. In some embodiments,the evolved product has increased or enhanced properties (e.g.,activity, specificity, stability, enantioselectivity) compared with theoriginal product of the gene of interest. In some embodiments, theevolved product has decreased or reduced properties (e.g., activity,specificity, stability, enantioselectivity) compared with the originalproduct of the gene of interest. Non-limiting examples of an evolvedproduct include modified T7 RNAP, modified RNA ligase ribozymes, betalactamase, modified zinc-finger binding domains, modified zinc-fingertargeted recombinases, and modified Tn3-family serine recombinaseenzymes, modified TEV protease, or modified single-chain variablefragments (scFvs).

In some embodiments, the evolved products have altered substratespecificity. In some embodiments, the evolved products have alteredactivity. In some embodiments, the evolved product resulting from acontinuous directed evolution experiment exhibits at least a 2-fold, atleast 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, atleast 40-fold, at least 50-fold, at least 60-fold, at least 80-fold, atleast 100-fold, or at least 120-fold greater activity than that of thestarting biomolecule used in to originally generate the evolved product.In some embodiments, the evolved product resulting from a continuousdirected evolution experiment exhibits at least 50-fold, at least100-fold, at least 500-fold, at least 1000-fold, at least 5000-fold, atleast 10,000-fold, at least 12,000-fold, or at least 15,000-fold changein specificity for its non-native target.

For example, some embodiments provide a modified T7 RNA polymerase (T7RNAP) having an altered substrate specificity and/or an increasedtranscriptional activity as compared to wild-type T7 RNAP. In someembodiments, the evolved product is a modified T7 RNAP that initiatestranscription from a T3 promoter. In some embodiments, the evolvedproduct is a modified T7 RNAP that initiates transcription from an SP6promoter. In some embodiments, the modified T7 RNAP exhibits an at least2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least30-fold, at least 40-fold, or at least 50-fold greater transcriptionrate from its native T7 promoter than the wild-type enzyme. In someembodiments, the modified T7 RNAP exhibits an at least 2-fold, at least5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least40-fold, at least 50-fold, at least 60-fold, at least 80-fold, at least100-fold, or at least 120-fold greater transcription rate from anon-native T7 RNAP promoter than the wild-type enzyme. In someembodiments, the non-native T7 RNAP promoter is a SP6 promoter. In someembodiments, the non-native T7 RNAP promoter is a T3 promoter. In someembodiments, the T7 RNA polymerase is at least about 1.5-fold, 2-fold,5-fold, 10-fold, 20-fold, 50-fold, 80-fold, 100-fold, 500-fold,800-fold, 1000-fold, 3,000-fold, 5,000-fold, 8,000-fold, 10,000-fold,12,000-fold, or 15,000-fold more specific for the P_(T3) over theP_(T7). In some embodiments, the T7 RNA polymerase is at least about1.5-fold to 10-fold, 10-fold to 50-fold, 50-fold to 80-fold, 80-fold to100-fold, 100-fold to 300-fold, 300-fold to 500-fold, 500-fold to800-fold, 1000-fold to 3,000-fold, 3,000-fold to 5,000-fold, 5,000-foldto 8,000-fold, 8,000-fold to 10,000-fold, 10,000-fold to 12,000-fold, or12,000-fold to 15,000-fold more specific for the P_(T3) over the P_(T7).

In some embodiments, the evolved product from a is a P_(T3)-specificRNAP that exhibits a net of at least 1000-fold, at least 5,000-fold, atleast 10,000-fold, or at least 15,000-fold-fold change in specificityfor P_(T3).

In some embodiments, the evolved product is generated using the methodsdescribed herein in at least about 1 hour, 2 hours, 4 hours, 6 hours, 8hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 24hours, 30 hours, 36 hours, 40 hours, 48 hours, 60 hours, 72 hours, or100 hours. In some embodiments the evolved product is generated in about2-12 hours, 12-24 hours, 24-48 hours, 48-60 hours, 60-72 hours, or72-100 hours.

In some embodiments, the evolved product is a T7 RNAP that evolved theability to accept the T3 promoter over the T7 promoter. In someembodiments, such evolved T7 RNAP has at least 1, 2, 3, 4, 5, or 6highly conserved mutations including those selected from R96L, K98R,E207K, E222K, N748D, and P759S/L. In some embodiments, the evolved T7RNAP is a N748D mutant; a E222K/N748D mutant; a E207K/E222K/N748Dmutant; a R96L/K98R/E207K/E222K/N748D mutant; aR96L/K98R/E207K/E222K/N748D/P759S mutant; or aR96L/K98R/E207K/E222K/N748D/P759L mutant.

The properties of an evolved product can be monitored over time using anumber of methods. For example, the phage population can be followedusing a combination of restriction endonuclease digests and/or real-timemeasurements of luminescence monitoring of promoter transcriptionalactivity (e.g., P_(T7) transcriptional activity), as further describedherein. Promoter transcriptional activity can be monitored by linkingthe activity to the expression of a reporter gene or proteinfluorescence. In some embodiments, an accessory plasmid carrying thegene III and a gene encoding a fluorescent protein (such as theluciferase gene, GFP, or other fluorescent protein described herein) areboth under the control of a conditional promoter (such as a P_(T7) orP_(T3)) and the expression of a fluorescent protein producesluminescence from the expressed luciferase when there is promotertranscriptional activity.

PACE Using Modulation of Selection Stringency and Negative Selection

Provided herein a method of using PACE with modulation of the selectionstringency followed by the combination of negative and positiveselection strategies to evolve a T7 RNAP with activity and specificityfor a T3 promoter.

In an embodiment, provided is a method for modulating the selectionstringency during phage-assisted evolution of a gene product, the methodcomprising: (a) introducing E. coli host cells from a chemostat into alagoon, wherein the host cell comprises a low selection stringencyplasmid and a high selection stringency plasmid, wherein the lowselection stringency plasmid comprises gene III wherein gene III isexpressed in response to the a concentration of anhydrotetracycline, andwherein the high selection stringency plasmid comprises a second copy ofgene III, wherein gene III is expressed in response to T3 RNAP activity;(b) introducing a selection phage comprising a gene encoding T7 RNAP tobe evolved into a flow of host cells through a lagoon; and (c) mutatingthe gene encoding T7 RNAP within the flow of host cells, wherein thehost cells are introduced through the lagoon at a flow rate that isfaster than the replication rate of the host cells and slower than thereplication rate of the virus thereby permitting replication of theselection viral vector in the lagoon.

In one embodiment, the low selection stringency plasmid contains aPpsp-tet drift promoter that is activated by a concentration ofanhydrotetracycline and prior viral infection. In one embodiment, thehigh selection stringency plasmid contains a T3 promoter that isactivated by a T3 RNAP or an T7 RNAP modified to have activity for a T3promoter. In yet another embodiment, the low selection stringencyplasmid comprises a mutagenesis cassette under the control of anarabinose inducible promoter. In another embodiment, the low selectionstringency plasmid allows a high level of evolutionary drift to occurwhen the Ppsp-tet drift promoter is activated in response to aconcentration anhydrotetracycline and prior viral infection. In oneembodiment, the high selection stringency plasmid allows a low level ofevolutionary drift to occur when the T3 promoter is activated inresponse to a T7 RNAP modified to have activity for a T3 promoter.

In one embodiment, the method of modulating the selection stringencyfurther comprises the use of negative selection and/or positiveselection.

In an embodiment, provided is a method of using negative selectionduring phage-assisted evolution of a gene product, the methodcomprising: (a) introducing E. coli host cells from a chemostat into alagoon, wherein the cell comprises a negative selection gene and adominant negative mutant gene encoding a pIIIneg protein, wherein thetranscription of the dominant negative gene is driven by a PT7 promoterin response to an undesired T7 RNAP activity of the gene to be evolvedor an evolution product thereof and the translation of the dominantnegative mutant gene encoding a pIII-neg protein is under the control ofa theophylline-inducible riboswitch, which is activated by aconcentration of theophylline; (b) introducing a mixture of selectionphage into a flow of host cells through a lagoon, wherein one type ofselection phage comprises a PT7 as the negative selection genecomprising an undesired T7 RNAP property of the T7 RNAP gene to beevolved and wherein another type of selection phage comprises a PT3 asthe positive selection gene comprising a desired T3 RNAP property of theT7 RNAP gene to be evolved; and (c) mutating the T7 RNAP gene to beevolved within the flow of host cells, wherein the host cells areintroduced through the lagoon at a flow rate that is faster than thereplication rate of the host cells and slower than the replication rateof the phage thereby permitting replication of the selection phage inthe lagoon. In one embodiment, the PT7 negative selection gene and geneIII-neg is found on an negative selection accessory plasmid. In anembodiment, the negative selection accessory plasmid further comprises asequence encoding for a riboswitch that is activated by a concentrationof theophylline. In one embodiment, the host cells further comprises amutagenesis cassette located on a mutagenesis plasmid and is undercontrol of an arabinose-inducible promoter. In one embodiment, thepIII-neg protein is the N-C83 variant.

In one embodiment, the method of negative selection further comprises amethod of positive selection, the method comprising a positive selectionaccessory plasmid comprising a gene that encodes for the pIII proteinand a gene encoding for luciferase, wherein the two are driven by the T3promoter. In an embodiment, the positive selection plasmid is differentfrom the negative selection plasmid.

In one embodiment, a new batch of host cell is subsequently fed into thelagoon from another chemostat, wherein the host cells comprise anegative selection accessory plasmid with a relatively stronger ribosomebinding site and an increased origin copy number and a positiveselection accessory plasmid comprising a reduced ribosome biding sitePT3-gene III. In an embodiment, the positive selection accessory plasmidfurther comprises a gene encoding for luciferase.

Apparatus for Continued Evolution

Apparatuses for continued evolution have been previously described.Provided herein is an apparatus comprising a chemostat. A chemostatcomprises a cell culture vessel in which the population of fresh hostcells is situated in liquid suspension culture. A constant nutrient flowis provided into the chemostat. Unlike a turbidostat, the turbidity in achemostat does not require monitoring. Like a turbidostat in previouslydescribed PACE apparatuses, the chemostat also comprises an outflow thatis connected to an inflow of the lagoon, allowing the introduction offresh cells from the chemostat into the lagoon.

In some embodiments, the media for the chemostat comprises a customDavis rich media formulation prepared from anhydrous K₂HPO₄ (140 g),KH₂PO₄ (40 g), (NH₄)₂SO₄ (20 g), NaCl (58 g), casamino acids (100 g),Tween-80 (20 mL), L-cysteine (1 g), L-tryptophan (0.5 g), adenine (0.5g), guanine (0.5 g), cytosine (0.5 g), uracil (0.5 g), CaCl2 (0.5 μMfinal). The media is allowed to cool overnight, and supplemented with a500 mL filter-sterilized solution of MilliQ water containing: NaHCO₃(16.8 g), glucose (90 g), sodium citrate (10 g), MgSO₄ (1 g), FeSO₄ (56mg), thiamine (134 mg), calcium pantothenate (94 mg), para-aminobenzoicacid (54 mg), para-hydroxybenzoic acid (54 mg), 2,3-dihydroxybenzoicacid (62 mg), (NH4)₆Mo₇ (3 nM final), H₃BO₃ (400 nM final), CoCl₂ (30nM), CuSO₄ (10 nM final), MnCl₂ (80 nM final), and ZnSO₄ (10 nM final).In an embodiment, cultures are supplemented with the appropriateantibiotics such as carbenicillin, spectinomycin, chloramphenicol,streptomycin, and/or tetracycline, depending on the antibiotic resistantgene found on the accessory plasmids in the host cells. In oneembodiment, the growth of the host cells is equilibrated overnight at aflow rate of about 100, 200, 300, 400, 500, or 800 mL/h. In oneembodiment, the growth of the host cells is equilibrated overnight at aflow rate of 400 mL/h. In an embodiment, the chemostat volume is about100 mL, 150 mL, 200 mL, 250 mL, 300 mL, 350 mL, 400 mL, 450 mL or 500mL. In an embodiment, the chemostat volume is about 250 mL.

Host Cells

Some aspects of this invention relate to host cells for continuousevolution processes as described herein. In some embodiments, a hostcell is provided that comprises at least one viral gene encoding aprotein required for the generation of infectious viral particles underthe control of a conditional promoter. For example, some embodimentsprovide host cells for phage-assisted continuous evolution processes,wherein the host cell comprises an accessory plasmid comprising a generequired for the generation of infectious phage particles, for example,M13 gIII, under the control of a conditional promoter, as describedherein. In some embodiments, the host cells comprise the gene requiredfor the generation of infectious viral particles under the control of aconditional promoter inserted into their genome, or as a cosmid,phagemid, or an artificial chromosome. In some embodiments, the hostcell is a bacterial cell, for example, a bacterial cell amenable to M13infection, such as certain E. coli cells. For M13 PACE, the host E. colicells need to express the F-factor, for example, from an F′ plasmid.Suitable F′ E. coli cell lines and strains are known to those of skillin the art, and include, for example, the F′proA⁺B⁺ Δ(lacIZY)zzf::Tn10(TetR)/endA1 recA1 galE15 galK16 nupG rpsL ΔlacIZYA araD139Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC) proBA::pir116λ⁻ cells describedherein.

In some embodiments, the host cell provided further comprises anexpression construct comprising a gene encoding a mutagenesis-inducingprotein, for example, a mutagenesis plasmid comprising a pBAD promoter,as described elsewhere herein.

Kits

Some aspects of this invention provide kits for continuous evolution asdescribed herein. In some embodiments, the kit comprise reagents,vectors, cells, software, systems, and/or apparatuses for carrying outthe methods provided herein. For example, in some embodiments, a kit forcontrolling the selection stringency in a continuous directed evolutionin a bacterial system is provided that includes a selection phage orphagemid; a drift plasmid; an accessory plasmid; optionally, amutagenesis plasmid and/or a mutagen; and/or a host cell capable ofproducing infectious phage and amenable to phage infection. In someembodiments, a kit is provided that comprises a two-plasmid PACE vectorsystem, as described in more detail elsewhere herein, for example,comprising a selection phage, a drift plasmid, an accessory plasmid,optionally, a mutagenesis plasmid, but no helper phage. In someembodiments, the kit further contains negative and/or positive selectionconstructs and optionally, a mutagenesis plasmid and/or a mutagen. Insome embodiments, the kit optionally contains small molecule inducers.The kit typically also includes instructions for its use.

The function and advantage of these and other embodiments of the presentinvention will be more fully understood from the Examples below. Thefollowing Examples are intended to illustrate the benefits of thepresent invention and to describe particular embodiments, but are notintended to exemplify the full scope of the invention. Accordingly, itwill be understood that the Examples are not meant to limit the scope ofthe invention.

EXAMPLES Example 1 Evolution of RNA Polymerase Variants Methods

Bacterial Strains.

All DNA cloning was performed with Mach1 cells (Invitrogen) or NEB Turbocells (New England Biolabs). All discrete infection assays, plaqueassays and PACE experiments were performed with E. coli S1030. Thisstrain was derived from E. coli S109¹ and was modified using the LambdaRed method²³ as follows: 1) scarless mutation to rpoZ to introduce aframeshift mutation to enable n-hybrid schemes;²⁴ 2) integration of lac′and tetR overexpression cassettes onto the F plasmid to enablesmall-molecule regulated transcription of various genes; 3) integrationof luxCDE onto the F plasmid for the production of decanal to facilitateluciferase monitoring experiments;²⁵ 4) deletion of flu, pgaC, andcsgABCDEFG to dramatically reduce biofilm formation in chemostat PACEexperiments;²⁶⁻²⁸ and 5) mutation of the chromosomal, low-affinityhigh-capacity AraE promoter to a constitutive promoter for titratablearabinose induction of the PBAD promoter on the mutagenesis plasmid.²⁹The complete genotype of the resulting strain is F′proA+B+ Δ(lacIZY)zzf::Tn10(TetR) lacIQ1 PN25-tetR luxCDE/endA1 recA1 galE15 galK16 nupGrpsL(StrR) ΔlacIZYA araD139 Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC)proBA::pir116 araE201 ΔrpoZ Δflu ΔcsgABCDEFG ΔpgaC λ−.

Plasmids.

A list of plasmids used in these examples are provided in Table 1.

TABLE 1 Antibiotic Origin of Name Class Resistance Replication PromoterGene pJC148i2 AP Carb SC101 P_(tet) gIII-luxAB pJC156a2 AP-neg SpectcolEI P_(lac) C-domain-Venus pJC156c2 AP-neg Spect colEI P_(lac)C83-Venus pJC156j2 AP-neg Spect colEI P_(lac) N1-N2-C83-Venus pJC156m2AP-neg Spect colEI P_(lac) N2-C83-Venus pJC156o2 AP-neg Spect colEIP_(lac) N1*-N2-C83-Venus pJC173b AP Carb SC101 P_(T7) gIII-luxAB pJC173cAP Carb SC101 P_(T7) 6xHisTag-gIII-luxAB pJC173e4 AP Carb SC101 P_(T7)luxAB pJC173f-R5 AP Spect colEI P_(T7) TheoRibo-6xHisTag-gIII- VenuspJC173g-SD8 AP-neg Spect pUC P_(T7) N1*-N2-C83-Venus (strong RBS)pJC174c-R5 AP Spect colEI P_(T3) TheoRibo-6xHisTag-gIII- Venus pJC174e4AP Carb SC101 P_(T3) luxAB pJC174f AP Carb SC101 P_(T3)6xHisTag-gIII-luxAB pJC174k AP Carb SC101 P_(T3) 6xHisTag-gIII-luxAB(weak RBS) pJC175e AP Carb SC101 P_(psp) gIII-luxAB pJC175g AP CarbSC101 P_(psp-tetO) gIII-luxAB pJC175h AP Carb SC101 P_(psp-tet)gIII-luxAB pJC175k AP Carb SC101 P_(psp-tetO3) gIII-luxAB pJC175m APCarb SC101 P_(psp-tetO5) gIII-luxAB pJC175n AP Carb SC101 P_(psp-tetO6)gIII-luxAB pJC184 MP Chlor CloDF13 P_(BAD) dnaQ926-umuD′-umuC- recA730pJC184c-rrnB AP Chlor CloDF13 P_(psp) gIII pJC184d5 DP Chlor CloDF13P_(BAD)/ dnaQ926-umuD′-umuC- P_(psp-tet) recA730/gIII pJC184d-Spect APChlor CloDF13 P_(psp-tet) gIII SP-T7_(WT) SP Kan f1 P_(gIII) wt T7 RNAPSP-T7_(SP) SP Kan f1 P_(gIII) T7 RNAP mutant L2-48.3 SP-T7_(Pr) SP Kanf1 P_(gIII) T7 RNAP mutant L1-192.2

Chemostat and Growth Media.

Compared to host cell cultures maintained at constant turbidity(turbidostats),¹ host cell cultures maintained at a constant nutrientflow rate (chemostats) are simpler to set up and do not require constantturbidity monitoring. We found that chemostats using E. coli S1030 cellsdescribed above support PACE comparably to turbidostats cultures, andtherefore used chemostats for the PACE experiments in this study.

Media for all chemostat PACE experiments was prepared by autoclaving a20 L carboy of MilliQ water containing a custom Davis rich mediaformulation: anhydrous K2HPO4 (140 g), KH2PO4 (40 g), (NH4)2SO4 (20 g),NaCl (58 g), casamino acids (100 g), Tween-80 (20 mL), L-cysteine (1 g),Ltryptophan (0.5 g), adenine (0.5 g), guanine (0.5 g), cytosine (0.5 g),uracil (0.5 g), CaCl2 (0.5 μM final). This was allowed to coolovernight, and supplemented with a 500 mL filter-sterilized solution ofMilliQ water containing: NaHCO3 (16.8 g), glucose (90 g), sodium citrate(10 g), MgSO4 (1 g), FeSO4 (56 mg), thiamine (134 mg), calciumpantothenate (94 mg), para-aminobenzoic acid (54 mg),para-hydroxybenzoic acid (54 mg), 2,3-dihydroxybenzoic acid (62 mg),(NH4)6Mo7 (3 nM final), H3BO3 (400 nM final), CoCl2 (30 nM), CuSO4 (10nM final), MnCl2 (80 nM final), and ZnSO4 (10 nM final). Cultures weresupplemented with the appropriate antibiotics in the following finalconcentrations: carbenicillin (AP; 50 μg/mL), spectinomycin (APneg; 50μg/mL), chloramphenicol (MP; 40 μg/mL). Streptomycin (for selection ofS1030 cells carrying the rpsL marker) and tetracycline (for selection ofthe F plasmid) were not routinely included in culture media.

Real-Time Luminescence Monitor.

The in-line bioluminescence measurements were made by a modifiedluminometer. A TD-20e luminometer (Turner Designs) was installed insidea dark box in continuous reading mode. While in this mode, theluminometer outputs a DC voltage that varies between 0 and 4 V dependingon the light level received by the photomultiplier fitted in theinstrument (the range was set to the most sensitive one). This signalwas sent to an Arduino Mega (Arduino) prototyping board through theanalog input port. The Arduino board was controlled via Matlab(Mathworks), using a custom Graphical User Interface (GUI). Measurementswere taken at around 100 Hz and then integrated to 1 s before beingwritten by the software into a text file. To allow for multichannelrecordings, the system was fitted with a custom tube holder that couldhold up to four flow-through tubes with minimal cross talk betweenchannels. Because the holder allows for only one tube to be observed ata time, it is mounted on a stepper motor that is in turn controlledusing the Matlab GUI via the Arduino board and a Pololu A4988 steppermotor driver (Pololu R&E). The system waits until a measurement iscomplete before rotating the sample holder until the next tube is inposition. The cycle repeats until all channels are covered, then unwindsto start a new cycle. The time between measurements of a particularchannel is ˜30 s. Data analysis comprised temporal binning and low-passFourier filtering of the raw traces.

Assay for Infection-Competence of Recipient Cells.

Recipient cultures were grown to mid-log phase, and 100 μL of culturewas mixed by pipetting with 2 μL of pJC126b phagemid prep (encodes R6Korigin, fl origin, pLac-YFP) totaling ˜109 cfu. Reactions were incubatedfor 2 min and then 1 μL of the reaction was diluted into 1 mL of fresh2×YT media. Cells were pelleted, resuspended in 1 mL fresh 2×YT, and analiquot was diluted 10-fold into fresh media. 20 μL of the resultingmedia was plated on 2×YT-agar plates with 1 mM IPTG and grown overnight.Colonies were scanned on a Typhoon laser imager (488 nm laser, 520/40filter).

Discrete Assay for Phage Production.

Cultures with candidate drift plasmids contained: P_(T7) (pJC173b), pTet(pJC148i2), psp (pJC175e), psp-tet (pJC175g/h/k/m/n), psp-tet on MP(pJC184d5), psp-tet on colE1-spect plasmid (pJC184d-Sp). Cultures weregrown to mid-log phase and infected with an excess of SP-T7. Cells werepelleted and washed to remove residual excess phage. Cells werere-inoculated into fresh media and grown to OD600˜0.8, and supernatantswere saved for titering. OD600 values were measured to normalize phagetiters.

Continuous Flow Experiment of PTet-Gene III Drift Cassette.

A chemostat culture of S1030 cells carrying an AP encoding PTet-gene III(pJC148i2) was prepared and growth was equilibrated overnight at a flowrate of 400 mL/h in a chemostat volume of 250 mL. The next morning, twolagoons (40 mL each, flow rate from the chemostat of 100 mL/h) wereseeded with 107 pfu of SP-T7. Lagoons received supplements delivered bysyringe pump (flow rate 1 mL/h) consisting of either 20 μg/mL ATc(resulting in 200 ng/mL final concentration) or water only. At 4-hourintervals, 0.6-mL samples were taken from lagoons, measured discretelyfor luminescence, and centrifuged to remove cells. Supernatants werecombined with an equal volume of 50% glycerol and stored in the freezer.Phage titers of supernatants were measured on S1030 cells containing anAP encoding P_(T7)-gene III (pJC173b).

Continuous Flow Experiment of Ppsp-Gene III Drift Cassette.

A chemostat culture of S1030 cells carrying an AP encoding Ppsp-gene III(pJC175e) was prepared and equilibrated overnight. The flow rate was 400mL/h in a chemostat volume of 250 mL. The next morning, a lagoon (40 mLeach, flow rate 100 mL/h) was started and seeded with 107 pfu ofSP-T7WT. At 4-hour intervals, 0.6-mL samples were taken from the lagoon,measured discretely for luminescence, and centrifuged to remove cells.Supernatants were combined with an equal volume of 50% glycerol andstored in the freezer. Phage titers of supernatants were measured onS1030 cells containing an AP encoding P_(T7)-gene III (pJC173b).

Continuous Flow Experiment of Ppsp-Tet-Gene III Drift Cassette.

A chemostat culture of S1030 cells carrying plasmid Ppsp-tet-gene III(pJC175h) was prepared equilibrated overnight. The flow rate was 400mL/h in a chemostat volume of 250 mL. The next morning, two lagoons (40mL each, flow rate 100 mL/h) were started and seeded with 107 pfu ofSP_(T7)WT. Lagoons received supplements delivered by syringe pump (flowrate 1 mL/h) consisting of either 20 μg/mL ATc (resulting in 200 ng/mLfinal concentration) or water only. At 4-hour intervals, 0.6-mL sampleswere taken from the lagoon, measured discretely for luminescence, andcentrifuged to remove cells. Supernatants were combined with an equalvolume of 50% glycerol and stored in the freezer. Phage titers ofsupernatants were measured on S1030 cells containing an AP encodingP_(T7)-gene III (pJC173b).

Continuous Flow Experiment of Ppsp-Tet-Gene III Drift Cassette with ATcDose-Dependent Stringency.

A chemostat culture of S1030 cells carrying a DP encoding Ppsp-tet-geneIII with a mutagenesis cassette (pJC184d5) and an AP encodingP_(T7)-6×HisTag-gene III (pJC173c) was prepared and equilibratedovernight. The flow rate was 400 mL/h in a chemostat volume of 250 mL.The next morning, three lagoons (40 mL each, flow rate 100 mL/h) werestarted, with their waste lines diverted through the in-lineluminescence monitor. Lagoons were seeded with a mixture containing 109pfu SP-T7Dead (D812G) and 107 pfu SP-T7WT, along with ATc in an amountthat brought the 40 mL lagoon to the target final ATc concentration foreach lagoon. Lagoons received supplements delivered by syringe pump(flow rate 1 mL/h) consisting of either 15 μg/mL ATc (to make 150 ng/mLfinal concentration), 40 μg/mL ATc (to make 400 ng/mL finalconcentration), or no ATc (water only). At t=8 h, the lagoon receiving40 μg/mL ATc was switched to receive water only. At timepoints t=2, 4,6, 8, and 16 h, 0.6-mL samples were taken from the lagoon, measuredindividually for luminescence, and centrifuged to remove cells.Supernatants were combined with an equal volume of 50% glycerol andstored in the freezer.

Supernatants were analyzed for relative phage ratios using a PCR andanalytical restriction digestion. 1 μL of each supernatant was added toa 20 μL qPCR reaction (iQ SYBR Green Mastermix, Biorad) with primersJC1480 (5′-TCCAGCACTTCTCCGCGATGCTC-3′, SEQ ID NO: XX) and JC1481(5′-GAAGTCCGACTCTAAGATGT CACGGAGGTTCAAG-3′, SEQ ID NO: XX) and amplifiedby PCR with SYBR Green fluorescence monitored following eachamplification cycle. Samples were removed individually from the PCRblock after exceeding a pre-determined fluorescence threshold, andplaced on ice. Samples were then quantitated again by SYBR Greenfluorescence in a plate reader, and approximately equal amounts of DNAfrom the reactions (normalized based on the fluorescence readings) wereadded to restriction digestion reactions. The digestion reactions usedenzymes EcoRV and HindIII in buffer NEB2 with BSA and were performedfollowing the manufacturer's instructions in 20 μL total volumes.Digestions were inactivated with heat, combined with 0.2 volumes of 5×loading dye containing Orange G and xylene cyanol, and analyzed byagarose gel electrophoresis (1% agarose, 0.5×TBE, 120V, 80 min). Theelectrophoresed gel was stained with SYBR Gold (Invitrogen) and imagedon a Typhoon laser scanner (excitation 488, emission 520/40 nm).

Continuous Flow Experiment of Dose-Dependent Negative Selection.

A chemostat of 51030 host cells containing an AP encoding P_(T7)-geneIII (pJC173c) and an APneg encoding P_(T3)-theophylline riboswitch-geneIII-neg (pJC174c-R5) was prepared and equilibrated overnight at a flowrate of 400 mL/h in a chemostat volume of 250 mL. The next morning,three lagoons (40 mL each, flow rate 100 mL/h) were started, with theirwaste lines diverted through the in-line luminescence monitor. Lagoonswere seeded with a mixture containing 1010 pfu SP-T7Prom and 104 pfuSP-T7Spec and allowed to equilibrate for 4.5 h; during this time thelagoons received 0.1 M NaOH at 0.5 mL/h by syringe pump to equilibratethe lagoons to the pH of the theophylline vehicle. After equilibration(t=0 h), lagoons received supplements delivered by syringe pump (flowrate 0.5 mL/h) consisting of either 200 mM theophylline (to make 1 mMfinal concentration), 15 mM theophylline (to make 75 μM finalconcentration), or vehicle only (0.1 M NaOH). After 16 h, the lagoonreceiving vehicle only was switched to receive 200 mM theophylline (1 mMfinal concentration). At timepoints t=0, 2, 4, 6, 8, 10, 12, and 20 h,0.6 mL-samples were taken from the lagoon, measured discretely forluminescence, and centrifuged to remove cells. Supernatants werecombined with an equal volume of 50% glycerol and stored in the freezer.

Relative phage ratios in supernatant samples were assayed using a PCRand analytical restriction digestion. 1 μL of each supernatant was addedto a 20 μL qPCR reaction (iQ SYBR Green Mastermix, Biorad) with primersJC1163 (5′-GGAGTACGCTGCATCGAGATGCTCA-3′, SEQ ID NO: XX) and JC1488(5′-GTAGAAATCAGCCAGTACATCACAAGACTC-3′, SEQ ID NO: XX) and amplified byPCR with SYBR Green fluorescence monitored following each amplificationcycle. Samples were removed individually from the PCR block afterexceeding a pre-determined fluorescence threshold, then placed on ice.Samples were quantitated again by SYBR Green fluorescence in a platereader, and approximately equal amounts of DNA from the reactions(normalized based on the fluorescence readings) were added torestriction digestion reactions. The digestion reactions used AvaII inbuffer NEB4 and were performed according to manufacturer instructions in20-μL total volumes. Digestions were combined with 0.2 volumes of 5×loading dye containing Orange G and xylene cyanol, and analyzed byagarose gel electrophoresis (1% agarose, 0.5×TBE, 120V, 80 min). Theelectrophoresed gel was stained with SYBR Gold (Invitrogen) and imagedon a Typhoon laser scanner (excitation 488, emission 520/40 nm).

Continuous Evolution of T3-Selective RNA Polymerases.

A chemostat culture of 51030 cells carrying a DP encoding Ppsp-tet-geneIII with a mutagenesis cassette (pJC184d5) and an AP encodingP_(T3)-gene III (pJC174f) was prepared and equilibrated overnight at aflow rate of 400 mL/h in a chemostat volume of 250 mL. The next morning,two lagoons (50 mL each, flow rate 100 mL/h) were started, with theirwaste lines diverted through the in-line luminescence monitor.

Lagoons were seeded with 105 pfu of SP-T7WT. Lagoons receivedsupplements delivered by syringe pump (flow rate 1 mL/h) consisting ofeither 20 μM ATc (in 500 mM L-arabinose) for lagoon 1 or vehicle alone(500 mM L-arabinose) for lagoon 2. At t=12 h, lagoon 1's supplement waschanged to 2 μM ATc (in 500 mM L-arabinose). At t=28 h, the firstchemostat was discontinued and both lagoons began receiving cells from asecond chemostat containing S1030 cells harboring an MP (pJC184), aP_(T3)-gene III AP (pJC174f), and a P_(T7)-theophylline riboswitch-geneIII-neg APneg (pJC173f-R5). Both lagoons received 500 mM L-arabinose. Att=32 h, half of lagoon 1 was transferred to a new lagoon, lagoon 3.Lagoons 1 and 3 were brought to 40 mL total volumes with culture fromthe chemostat, and lagoons were equilibrated for 40 min. Lagoons 1 and 2then received 100 mM theophylline (dissolved in 0.1 M NaOH, flow rate 1mL/h), while lagoon 3 received vehicle only (0.1 M NaOH). At t=52 h, thesecond chemostat was discontinued and all lagoons began receiving cellsfrom a third chemostat containing S1030 cells harboring an MP (pJC184),a reduced RBS P_(T3)-gene III (pJC174k) and an enhanced RBS/copy numberP_(T7)-gene III-neg APneg (pJC173g-SD8). All lagoon volumes were reducedto 40 mL each (flow rate maintained at 100 mL/h) and were supplementedwith 500 mM L-arabinose. At t=70.5 h, lagoon volumes were reduced to 30mL each, while the flow rate was maintained at 100 mL/h.

Periodically, 0.6 mL-samples were taken from the lagoon, measureddiscretely for luminescence, and centrifuged to remove cells.Supernatants were combined with an equal volume of 50% glycerol andstored in the freezer.

In Vivo Gene Expression Measurements on Evolved RNA Polymerases.

Cells for luminescence assays were S1030 cells described abovecontaining APs encoding Ppspgene III (pJC184c-rrnB) and eitherP_(T7)-luxAB (pJC173e4) or P_(T3)-luxAB (pJC174e4). Cells were grown tomid-log phase and 20-μL aliquots were distributed into a deep-wellplate. 10 μL of clonal phage aliquots were added to these wells andplates were incubated at 37° C. for 15 min. Wells were supplemented with500 μL media, grown to mid-log phase, and cells were transferred to a96-well plate for luminescence measurement.

In Vivo Gene Expression Measurements on RNA Polymerase Forward Mutants.

Cells for luminescence assays were S1030 cells described abovecontaining an expression plasmid (EP) expressing a subcloned forwardmutant T7 RNAP and APs encoding either P_(T7)-luxAB (pJC173e4) orP_(T3)-luxAB (pJC174e4). Cultures were grown overnight and used to seed500-μL cultures in 96-well blocks that were grown to mid-log phase,after which cells were transferred to a 96-well plate for luminescencemeasurement.

Discrete Assays for Phage Production from Candidate Drift Promoters.

Cultures of S1030 E. coli cells with candidate drift plasmids containingP_(T7)-gene III (pJC173b), PTet-gene III (pJC148i2), Ppsp-gene III(pJC175e), Ppsp-tet-gene III (pJC175g/h/k/m/n), Ppsp-tet-gene III on MP(pJC184d5), or Ppsp-tet-gene III on colE1-spect plasmid (pJC184d-Spect)were prepared and grown to mid-log phase in Davis rich media. Cultureswere infected with an excess of SP-T7WT and incubated at 37° C. for 10min. The resulting cells were pelleted and washed to remove residualexcess phage. These cells were inoculated into fresh 2×YT media andgrown to OD600=˜0.8. Cells were removed by centrifugation. Phagesupernatant was harvested from these cultures and used for titering byplaque assay on S1030 E. coli cells carrying a P_(T7)-gene III AP(pJC173b). Exact ODs were used to normalize phage titers.

Discrete Assays for Phage Production from pIII-Neg Candidates.

Cultures of S1030 E. coli cells with candidate pIII-neg plasmidscontaining PTet-gene III (pJC148i2) and either Plac-C-domain (pJC156a2),Plac-C83 (pJC156c2), Plac-N1-N2-C83 (pJC156j2), Plac-N2-C83 (pJC156m2),or Plac-N1*-N2-C83 (pJC156o2) were prepared and grown to mid-log phasein Davis rich media. Cultures were infected with an excess of SP-T7WTand incubated at 37° C. for 10 min. The resulting cells were pelletedand washed to remove residual excess phage. These cells were inoculatedinto fresh 2×YT media and grown in either 4 ng/mL or 20 ng/mL ATc and inthe presence or absence of 2 mM IPTG. Cultures were grown to OD600=˜0.9.Cells were removed by centrifugation. Phage supernatant was harvestedfrom these cultures and used for titering by plaque assay on S1030 E.coli cells carrying a P_(T7)-gene III AP (pJC173b). Exact ODs were usedto normalize phage titers.

Discrete Assays for Phage Production Using the Theophylline-DependentNegative Selection.

A culture of S1030 E. coli cells containing P_(T7)-gene III (pJC173c)and P_(T3)-theophylline riboswitch-gene III-neg (pJC174c-R5) wereprepared and grown to mid-log phase in Davis rich media. Cultures wereinfected with an excess of either SP-T7192.2 or SP-T748.3 and incubatedat 37° C. for 10 min. The resulting cells were pelleted and washed toremove residual excess phage. These cells were inoculated into fresh2×YT media and grown with the indicated concentrations of theophylline.Cultures were grown to OD600=˜0.8. Cells were removed by centrifugation.Phage supernatant was harvested from these cultures and used fortitering by plaque assay on S1030 E. coli cells carrying a P_(T7)-geneIII AP (pJC173b). Exact ODs were used to normalize phage titers.

Results

We hypothesized that selection stringency during PACE can be varied byproviding host cells with regulated amounts of pIII in a mannerindependent of the desired evolving activity. To create this capability,we placed expression of gene III on an AP under the control of the smallmolecule-inducible TetA promoter (Ptet) and observed anhydrotetracycline(ATc) concentration-dependent gene III expression (FIG. 2). Although anAP containing Ptet-gene III supported ATc-dependent phage production ina discrete host cell culture (FIG. 2), this AP did not support robustphage propagation in a PACE format when ATc was added to a lagoon (FIG.1c ). Because even low levels of pIII render cells resistant tofilamentous phage infection,¹⁰ we hypothesized that these host cells,which begin producing pIII soon after entering a lagoon, become phageinfection-resistant prior to encountering phage, thereby preventingphage propagation (FIG. 2).

To create a system in which activity-independent pIII expressionrequires both ATc and prior phage infection, we used the previouslydescribed E. coli phage shock promoter (Ppsp), which is induced byinfection with filamentous phage via a pIV-dependent signalingcascade.¹¹ Transcription from Ppsp can also be induced by overexpressionof a plasmid-encoded phage pIV gene.¹² Phage lacking gene III are knownto form plaques on cells containing a Ppsp-gene III cassette.¹³ Weconfirmed and extended this observation by showing that an AP with thiscassette supports robust propagation in a PACE format (FIG. 1d ).

To make phage propagation ATc-dependent, we examined the gene expressionproperties of Ppsp variants with TetR operators installed at positionsintended to disrupt either PspF or E. coli RNA polymerase binding (FIG.2). We found that placing an operator adjacent to the +1 transcriptioninitiation site creates a promoter (Ppsp-tet) that is induced only withthe combination of phage infection and ATc (FIG. 2). This AP supportedrobust ATc-dependent propagation of a SP with activity-independent geneIII expression (FIG. 1e ). High Ppsp-tet-gene III induction (200 ng/mLATc) supported propagation with dilution rates of 2.5 vol/h,corresponding to an average of 30 phage generations per 24 hours. Theseresults collectively demonstrate that SPs can propagate in anATc-dependent, activity-independent manner using the Ppsp-tet-gene IIIAP, thus enabling selection stringency to be altered during PACE in asmall molecule-regulated manner.

Tuning Selection Stringency During PACE.

Next we examined how the Ppsp-tet-gene III cassette influences theenrichment of active library members over inactive library members inthe context of an actual PACE selection in which an additional copy ofgene III is controlled by an activity-dependent promoter. At saturatingconcentrations of ATc, the Ppsp-tet-gene III cassette should providesufficient pIII to maximize phage propagation regardless of SP encodedactivity, enabling genetic drift of the SP. At intermediateconcentrations of ATc, SPs encoding active library members should enjoya replicative advantage over a SP encoding an inactive variant byinducing additional pIII expression from an activity-dependent promoter.This advantage, and therefore selection stringency, should be inverselyproportional to the concentration of ATc provided. In the absence ofadded ATc, selection stringency should be determined by theactivity-dependent AP components with no assistance from thePpsp-tet-gene III cassette.

To characterize the relationship between ATc concentration and selectionstringency, we combined the Ppsp-tet-gene III cassette with thearabinose-inducible mutagenesis cassette¹ onto a single “drift plasmid”(DP) that is compatible with activity-dependent APs. We set up lagoonswith continuously flowing host cells that contained both this DP and anAP with a P_(T7)-gene III cassette (FIG. 3a ). We then seeded lagoonswith a mixture of two SPs encoding either the highly active wild-type T7RNAP (SP-T7WT) or the catalytically inactive mutant D812G (SP-T7Dead).¹⁴These phage were added in a ratio of 1:100 SP-T7WT:SP-T7Dead to each ofthree lagoons receiving either 0, 150, or 400 ng/mL ATc. The phagepopulations were followed over time using a combination of restrictionendonuclease digests and real-time measurements of luminescencemonitoring of P_(T7) transcriptional activity.

As shown in FIG. 3b , under the highest stringency conditions (0 ng/mLATc), SP-T7Dead washed out of the lagoon and the highly active SP-T7WTwas enriched. At an intermediate concentration of ATc (150 ng/mL),SP-T7Dead again washed out in favor of SP-T7WT, but at a slower ratethan in the absence of ATc (FIG. 3c ). At the highest concentration ofATc (400 ng/mL), SP-T7Dead was able to propagate and no substantialenrichment of the active SP-T7WT was observed (FIG. 3d ). When westopped ATc supplementation to this lagoon, we observed very rapidenrichment of SP-T7WT consistent with the expected increase instringency (FIG. 3d ). Taken together, these results establish thatPpsp-tet-gene III supports propagation of inactive starting libraries inthe presence of high ATc concentrations, and supports the selectiveenrichment of active mutants at a rate inversely proportional to theconcentration of ATc added to the lagoon.

Development of a PACE Negative Selection.

An ideal PACE negative selection inhibits infectious phage production ina manner that is tunable and proportional to the ratio of undesired(off-target) to desired (on-target) activity, rather than simplyreflecting the absolute level of undesired activity. We therefore soughtto develop a negative selection in which undesired activity inducesexpression of a protein that antagonizes the wild-type pIII induced in apositive selection.

The pIII protein consists of three domains, N1, N2, and C, that mediateinitial attachment of the phage to the E. coli F-pilus (N2 domain),subsequent docking with the E. coli TolA cell-surface receptor (N1domain), and unlocking of the particle for genome entry (Cdomain).^(15,16) During progeny phage synthesis, five pIII molecules areattached by the C-domain to the end of each nascent phageparticle.^(17,18) A presumed conformational change in this C-domain thencatalyzes detachment of the nascent phage from the inner membrane,thereby releasing the phage from the host cell.¹⁶

One pIII mutant described by Rakonjac and co-workers, N-C83, containsintact N1 and N2 domains but has a mutant C domain with an internaldeletion of 70 amino acids.¹⁶ The residual C domain is sufficient tomediate attachment to a phage particle, but its ability to catalyzedetachment of nascent phage from the host cell membrane is hindered.¹⁶When co-expressed with a pIII variant containing only the intact Cdomain (which can mediate nascent phage detachment), phage particleproduction was found to be normal, but the infectivity of theseparticles was reduced.¹⁶ These observations raised the possibility thatN-C83 may function as a dominant negative mutant of wild-type pIII.

To test the suitability of N-C83 as the basis for a PACE negativeselection, we created a host cell line containing an AP with a Ptet-geneIII cassette induced by ATc, a second accessory plasmid (APneg) withN-C83 driven by an IPTG-inducible promoter (Plac). Discrete host cellcultures were grown in the presence or absence of ATc and IPTG, andculture broths were assayed for infectious phage titer. As expected, theaddition of ATc induced expression of wild-type pIII and stimulatedphage production (FIG. 4). The simultaneous addition of IPTG, whichinduces N-C83 expression, reduced the titer of infectious phage. Thisresult suggests that N-C83 can act as a dominant negative form of pIII.Alternative truncation candidates of pIII did not diminish infectiousphage titer to the same degree as N-C83 (FIG. 4). We also observedevidence to suggest that N-C83 may inhibit infectious phage productionby blocking the release of phage from the host cell, rather than byreducing the infectivity of the released phage particles (FIG. 6). Theseresults reveal that expression of N-C83 (pIII-neg), can form the basisof a potent negative selection.

PACE Negative Selection can Enrich Substrate-Specific RNAP Variants.

Next we tested if this negative selection could enrich asubstrate-specific T7 RNAP over a promiscuous RNAP in the PACE format.For this competition experiment, we used SPs encoding two RNAP variantsthat we recently described: L2-48.3 (specific for P_(T7), expressed fromSP-T7Spec) and L1-192.2 (active on both P_(T7) and P_(T3), expressedfrom SP-T7Prom).¹ We prepared a host cell strain containing both apositive selection AP (P_(T7)-gene III AP) and a negative selection AP(P_(T3)-gene III-neg APneg) in which pIII-neg translation is controlledby a theophylline-activated riboswitch (FIG. 7a ).¹⁹ During PACE withthis host cell strain, the propagation of SP-T7Prom should be impaired,relative to SP-T7Spec, in the presence of theophylline.

Three lagoons containing these host cells were each seeded with a 10⁶:1ratio of SP-T7Prom:SP_(T7)Spec and allowed to equilibrate for about 4.5hours. We treated the lagoons with 0, 75, or 1000 μM theophylline andfollowed the concentrations of the two SP species over time. In thepresence of 1000 μM theophylline, SP-T7Prom was rapidly depleted fromthe lagoon and the SP-T7Spec population expanded into the predominantspecies after about 6 hours of PACE (FIG. 7b ). Furthermore, the rate ofenrichment was dependent on the concentration of theophylline added tothe lagoon, where lower concentrations of theophylline resulted inslower enrichment rates (FIGS. 7c and 7d ). These results demonstratethat pIII-neg can serve as the basis of a potent and dose-dependentnegative selection that can effect the rapid enrichment ofsubstrate-selective enzymes in a PACE format.

Continuous Evolution of RNAPs with Altered Promoter Specificities.

We integrated both the tunable selection stringency and the negativeselection developments described above to rapidly evolve enzymes withdramatically altered substrate specificity without the use ofintermediate substrates. We prepared two lagoons with host cellscontaining a P_(T3)-gene III AP and the DP containing the Ppsp-tet-geneIII cassette, and seeded these lagoons with SP-T7WT, which hasnegligible starting activity on P_(T3). To one lagoon, we added no ATcand observed that phage quickly washed out.¹ To the second lagoon, weadded 200 ng/mL ATc, which allowed even inactive phage to propagate.After 12 hours (t=12 h), the phage in this lagoon increased inconcentration dramatically, but still encoded RNAPs with negligibleactivity on P_(T3). We then reduced the concentration of ATc to 20ng/mL, thus increasing the selection stringency for recognition ofP_(T3) (FIG. 8).

After an additional six hours of propagation in 20 ng/mL ATc (t=18 h), aP_(T3)-active population overtook the lagoon as indicated by an increasein signal from the in-line luminescence monitor. This signal continuedto increase over the next 10 hours. RNAP clones isolated from the28-hour time point evolved an ˜100-fold increase in activity on P_(T3)(FIG. 9) and converged on two mutations, E222K and N748D, that we¹ andothers²⁰ previously found to broaden the substrate scope of T7 RNAP(FIG. 10). These results demonstrate that selection stringencymodulation can be used to directly evolve a novel activity from aninactive starting gene without the use of intermediate substrates asevolutionary stepping-stones, as was previously used to evolve T3promoter recognition without selection stringency modulation.¹

As shown in FIG. 9, the RNAPs recovered from this stage of the selectionstill retained high activity on P_(T7). To initiate negative selectionagainst recognition of P_(T7), we used host cells containing the sameP_(T3)-gene III AP, the MP (lacking the drift cassette), and aP_(T7)-gene III-neg APneg in which pIII-neg expression is driven byP_(T7) transcription, and controlled by a theophylline-dependentriboswitch (FIG. 8). The 28-hour lagoon described above was divided intotwo new lagoons, each of which received these host cells for 4 hours.Theophylline was added to the first lagoon to activate the negativeselection (t=32 h), whereas the second lagoon did not receive anytheophylline. After 24 hours (t=52 h), several RNAPs isolated from thefirst lagoon showed clear improvements in specificity for P_(T3) (FIG.9) and contained a variety of mutations at residues proximal to thepromoter in the initiation complex and known to be relevant to substrateselectivity. In contrast, clones isolated from the second lagoon, whichdid not receive theophylline, did not show clear changes in specificity.

In an effort to further enhance the specificity of the evolving RNAPs,we increased the stringency of the negative selection in two ways.First, we weakened the ribosome binding site driving translation of pIIIfrom the AP, with the expectation that evolved RNAPs would compensatewith increased (but still P_(T3)-selective) transcriptional activity.Second, we installed a high-copy pUC origin into APneg, and replaced thetheophylline riboswitch with a very strong ribosome-binding site drivingpIII-neg translation (FIG. 8). Together, these modifications shouldincrease the stringency of the negative selection by increasing theratio of pIII-neg to pIII produced for a given ratio of P_(T7):P_(T3)activity.

The phage population from the previous stage (t=52 h) was transferred tolagoons fed by these high-stringency host cells and a substantialdecline and rebound of the phage population was observed (FIG. 8).Evolved RNAPs isolated following this recovery (t=70 h) exhibiteddramatically improved specificity for P_(T3) over P_(T7) of up to100-fold and strongly converged on mutations observed in the previousstage (R96L, K98R, E207K, P759S/L) (FIGS. 9 and 10). A forwardmutational analysis of these mutations in the E222K/N748D backgroundhighlighted the role of each of these changes in conferring strongspecificity for P_(T3) (FIG. 11). The degree of specificity evolvedusing stringency control and negative selection PACE rivals or exceedsthat of naturally occurring wild-type T7 or T3 RNA polymerase enzymes;for example, wild-type T7 RNAP exhibits a P_(T7):P_(T3) activity ratioof ˜100-fold, and wild-type T3 RNAP exhibits a P_(T3):P_(T7) activityratio of ˜20-fold (FIG. 11). Compared to the wild-type T7 RNAP enzyme,the most P_(T3)-specific evolved RNAP clones exhibited a net˜10,000-fold change in specificity for P_(T3). This remarkable degree ofspecificity did not evolve at the expense of activity, and all assayedclones retained levels of transcriptional activity on P_(T3) that arecomparable to or higher than that of wild-type T7 RNAP on its cognate T7promoter (FIG. 9). Collectively, these results establish that the PACEnegative selection can be used to explicitly evolve enzymes with alteredspecificity.

Discussion

Traditional directed evolution techniques, while valuable andsuccessful, require frequent researcher intervention throughout eachround of mutation, screening or selection, and replication to accessbiomolecules with desired properties. By mapping the essentialcomponents of the directed evolution cycle onto the very rapid M13filamentous bacteriophage lifecycle, PACE can dramatically acceleratelaboratory evolution. In this work we have expanded the capabilities ofPACE by developing a general strategy to modulate selectionstringency—to zero if needed—with a small molecule, and by developing anegative selection that links undesired activities to the inhibition ofphage propagation. Together, these capabilities were used to evolve RNApolymerase variants with ˜10,000-fold altered promoter specificity(rather than merely broadened), in a PACE experiment lasting about threedays.

T7 RNA polymerases that evolved the ability to accept the T3 promoterand reject the T7 promoter acquired a suite of highly conservedmutations, including R96L, K98R, E207K, E222K, N748D, and P759S/L. Themutations at positions 98 and 748 change each residue to the amino acidfound in wild-type T3 RNAP, and the R96L mutation is at a position thatalso differs between wild-type T7 and T3 RNAPs (although it is K96 in T3RNAP). These residues are predicted to be proximal to the promoter bases(FIG. 11b ), and their role in promoter recognition has been previouslyappreciated.²¹ The occurrence of these mutations in our selectionssuggests that the evolved mutants may use a mode of P_(T3)-selectivepromoter recognition similar to that of wild-type T3 RNAP at thesepositions. In contrast, residues 207, 222, and 759 are conserved betweenT7 and T3 RNAPs, and the significant role of mutations at these residuesin conferring selectivity (particularly P759S/L, FIG. 11a ) suggests theevolution of some novel determinants of promoter recognition that maynot be used by the native enzymes. E207 modestly increases selectivityfor P_(T3) (FIG. 11a ) and is within 4 Å of the RNAP specificity loop(residues 739-770), a major determinant of promoter specificity²² thatincludes P759.

The negative selection uses a dominant negative variant of pIII, theprotein that is the basis of the positive selection of PACE, and doesnot rely on the property of the gene or gene product being evolved.Therefore, we anticipate that the negative selection will be general inits compatibility with a variety of activities that can be evolved usingPACE, provided that the selection scheme can link undesired activity toexpression of pIII-neg (and not pIII). For many potential proteins thatcan be evolved with PACE, including DNA-binding proteins, recombinases,protein-protein interfaces, proteases, and other enzymes, a suitabledual selection scheme can be created by localizing undesired substratesupstream of pIII-neg production.¹ In most cases, such a negativeselection can operate simultaneously with pIII-mediated positiveselection. Likewise, since the method to modulate selection stringencydeveloped here is affected by providing pIII in a regulated manner thatis also independent of the evolving gene, this stringency modulationcapability should also be applicable to other PACE experiments.

Example 2 Development of a PACE System for DNA-Binding Activity

Engineered proteins containing programmable DNA-binding domains (DBDs)can be used for targeted modification of nucleic acid molecules in vitroand in vivo and have the potential to become human therapeutics. Thespecificity of a DNA-binding domain is crucial to the efficacy andsafety of the resulting DNA-binding proteins, such as nucleases orDNA-editing proteins. This disclosure illustrates a general approach forthe continuous directed evolution of DNA-binding activity andspecificity.

A general system for the continuous evolution of DNA-binding domains ispresented. The system was validated by evolving restored DNA-bindingactivity in zinc fingers. The data presented here establish a newstrategy for tuning the affinity and specificity of DBDs.

Results

To develop a PACE-compatible DNA-binding selection, a DNA-binding domainof interest was linked to a subunit of bacterial RNA polymerase III(RNAP). Binding of this fusion protein to operator sequences upstream ofa minimal lac promoter induces transcription of a downstream geneIII-luciferase reporter through recruitment or stabilization of the RNAPholoenzyme (FIG. 12, upper panel). To validate this strategy, an assaywas developed that transduces cognate DNA-binding of the DBD from Zif268(residues 333-420, see Choo, Y. & Klug, A. Toward a code for theinteractions of zinc fingers with DNA: selection of randomized fingersdisplayed on phage. Proc Natl Acad Sci USA 91, 11163-7 (1994).),expressed from a tetracycline-inducible promoter, into activation ofpIII-luciferase expression. This assay was used to evaluate a variety ofDNA operator locations (at −55 and −62 bp with respect to thetranscription initiation site, see Hu, J. C., Kornacker, M. G. &Hochschild, A. Escherichia coli one- and two-hybrid systems for theanalysis and identification of protein-protein interactions. Methods 20,80-94 (2000); and Durai, S., Bosley, A., Abulencia, A. B.,Chandrasegaran, S. & Ostermeier, M. A bacterial one-hybrid selectionsystem for interrogating zinc finger-DNA interactions. Comb Chem HighThroughput Screen 9, 301-11 (2006).) and RNA polymerase fusionarchitectures.

Fusing the RNAP w-subunit to the N-terminus of Zif268 with an 11-residuelinker resulted in ≧10-fold increase in pIII-luciferase production whenthe consensus Zif268 binding site (5′-GCGTGGGCG-3′) was positioned at−62 (FIG. 13). To test the DNA specificity of this system, a controlconstruct was created with an off-target Zif268 binding site in whichthe middle triplet of the target DNA site was changed to 5′-TTA-3′. OnlyE. coli containing the reporter downstream of the on-target sequence,but not those containing the off-target sequence, producedpIII-luciferase (FIG. 12, middle panel), establishing sequence-specific,DNA binding-dependent gene expression.

A series of negative selection APs (APNegs) was designed in whichbinding of a DBD to an off-target DNA sequence induces expression ofgene III-neg (encoding pIII-neg) fused to yellow fluorescent protein(YFP) from a minimal lac promoter (FIG. 12, lower panel). To enabletuning of negative selection stringency, a theophylline-inducibleriboswitch was placed upstream of gene III-neg-YFP. It was confirmedthat phage propagation could be suppressed in a DNA-binding activity-and theophylline-dependent manner. Together, these results establish anegative selection system for DNA-binding PACE.

To integrate this system into PACE, the positive DNA operator-geneIII-luciferase cassette and the negative DNA operator-gene IIIneg-YFPcassette was moved to accessory plasmids (APs), and the RNAP ω-Zif268protein was moved to a selection plasmid (SP).

An E. coli strain designated S2060 was developed that is capable ofinducing LacZ in response to activation of the phage shock promoter, atranscriptional regulatory element that responds to a number ofenvironmental signals including filamentous phage infection (FIGS.14-16). This strain can be used in combination with colorimetric LacZsubstrates such as X-gal to stain bacteria that have been infected withphage.

It was tested whether ω-Zif268-SP could propagate in a DNA bindingactivity-dependent manner on S2060 cells containing an AP with thecognate Zif268 binding sequence, or a mutated binding sequence. Robustformation of colored plaques was observed, indicative of phagepropagation, on cells harboring the on-target AP, but not on cellsharboring an AP containing the off-target sequence (FIG. 17a ). Theseobservations demonstrate DNA binding activity-dependent phagepropagation.

An initial PACE experiment was performed to optimize the SP backbone.SPs encoding Zif268 were propagated in PACE over 24 h on host cellscarrying the cognate AP plasmid and a mutagenesis plasmid (MP, seeEsvelt, K. M., Carlson, J. C. & Liu, D. R. A system for the continuousdirected evolution of biomolecules. Nature 472, 499-503 (2011). After 24h of PACE, the surviving SPs contained mutations in the phage genesencoding pII/X and pIV, and the fusion protein linker (FIG. 17b ). Theseresults collectively establish a basis for the continuous evolution ofDBDs using PACE.

To validate the ability of this positive selection PACE system toimprove DNA-binding activity, the system was used to evolve DNA bindingin an inactive Zif268 mutant protein. Mutation of Arg24 in Zif268 to asmall hydrophobic residue is known to abrogate DNA binding(Elrod-Erickson, M. & Pabo, C. O. Binding studies with mutants ofZif268. Contribution of individual side chains to binding affinity andspecificity in the Zif268 zinc finger-DNA complex. J Biol Chem 274,19281-5 (1999).). A lagoon was seeded with inactive ω-Zif268 SPcontaining an R24V mutation. After 24 h of neutral drift (mutation inthe absence of any selection pressure) followed by 24 h of PACE on hostcells containing the cognate AP, the evolved SPs were capable ofpropagating on the target AP (FIG. 17c ). All of the sequenced phageclones at the end of the 24-h PACE experiment contained the V24Rreversion mutation using an Arg codon not present in the wild-type gene(AGA vs. CGC) (FIG. 17d ). Collectively, these results validate thatthis system can rapidly evolve proteins with DNA-binding activity.

The DNA-binding PACE system developed in this work can be used torapidly tune the activity and specificity of a variety of DNA bindingproteins. A distinguishing feature of DNA-binding PACE is that it doesnot require the use of pre-defined selection libraries that canconstrain or bias evolutionary outcomes. The unconstrained manner inwhich mutations arise during PACE facilitates the discovery of evolvedsolutions with desired properties that could not be rationalized apriori.

Materials and Methods

Phage-Assisted Continuous Evolution (PACE) of DNA-Binding Domains.

In general, PACE setup was performed as previously described (Carlson,J. C., Badran, A. H., Guggiana-Nilo, D. A. & Liu, D. R. Negativeselection and stringency modulation in phage-assisted continuousevolution. Nat Chem Biol 10, 216-22 (2014)). E. coli were maintained inchemostats containing 200 mL of Davis' Rich Media (DRM) using typicalflow rates of 1-1.5 vol/h. DRM media was supplemented with appropriateantibiotics to select for transformed plasmids: APs (50 μg/mLcarbenicillin), APNegs (75 μg/mL spectinomycin), MPs (25 μg/mLchloramphenicol). Lagoon dilution rates were 1.3-2 vol/h. In all PACEexperiments S1030 cells carried an MP, either the previously reportedpJC184 (Carlson et al., 2014, see above), or a variant of this plasmidlacking RecA. Mutagenesis was induced by continuously injectingarabinose (500 mM) at a rate of 1 mL/h into each 40-mL lagoon. Typicalphage titers during each PACE experiment were 10⁶-10⁸ p.f.u./mL.

Reversion of Zif268-V24R.

A lagoon receiving host cell culture from a chemostat containing S1059cells transformed with an MP was inoculated with Zif268-V24R phage. Thelagoon flow rate during drift was 2 vol/h. After 24 h of drift, phagewere isolated and used to inoculate a PACE experiment with S1030 hostcells carrying pAPZif268 and an MP. Evolved phage were isolated after 24h and characterized using plaque assays.

Luciferase Assay.

pOH plasmids were transformed by electroporation into S1030 cells, andgrown overnight at 37° C. on LB-agar plates supplemented with 50 μg/mLcarbenicillin. Single colonies were used to inoculate cultures whichwere allowed to grow for ˜12 h at 37° C. in DRM supplemented with 50μg/mL carbenicillin in a shaker. Cultures were diluted to an OD₆₀₀ of˜0.3 and allowed to grow for an additional 2 h at 37° C. Next, eachculture was diluted 1:15 into 300 μL of DRM supplemented with 50 μg/mLcarbenicillin in the presence or absence of 200 ng/mLanhydrotetracycline and incubated in a 96-well plate for an additional4-6 h (shaking). 200 μL aliquots of each sample were then transferred to96-well opaque plates and luminescence and OD₆₀₀ readings were takenusing a Tecan Infinite Pro instrument. Luminescence data were normalizedto cell density by dividing by the OD₆₀₀ value.

Plaque Assays.

S1030 cells were transformed with the appropriate plasmids viaelectroporation and grown in LB media to an OD₆₀₀ of 0.8-1.0. Dilutedphage stock samples were prepared (10⁻⁴, 10⁻⁵, 10⁻⁶, or 10⁻⁷-folddilution) by adding purified phage stock to 250 μL of cells in Eppendorftubes. Next, 750 μL of warm top agar (0.75% agar in LB, maintained at55° C. until use) was added to each tube. Following mixing by pipette,each 1 mL mixture was pipetted onto one quadrant of a quartered petriplate that had previously been prepared with 2 mL of bottom agar (1.5%agar in LB). Following solidification of the top agar, plates wereincubated overnight at 37° C. prior to analysis. Colorimetric plaqueassays were performed in parallel with regular plaque assays using S2060cells instead of S1030 cells, and used S-Gal/LB agar blend (Sigma) inplace of regular LB-agar.

YFP Assays.

pTet plasmids were co-transformed with pAPNeg plasmids byelectroporation into S1030 cells, and grown overnight at 37° C. onLB-agar plates supplemented with 50 μg/mL carbenicillin and 100 μg/mLspectinomycin. Single colonies were used to inoculate cultures whichwere allowed to grow for ˜12 h in antibiotic-supplemented DRM in abacterial shaker. Cultures were diluted to an OD₆₀₀ of ˜0.3 and allowedto grow for an additional 2 h at 37° C. Next, each culture was diluted1:15 into 300 μL of DRM supplemented with antibiotics and 5 mMtheophylline in the presence or absence of 50 ng/mL anhydrotetracyclineand incubated in a 96-well deep well plate for an additional 4-6 h(shaking). 200 μL aliquots of each sample were then transferred to96-well opaque plates and YFP fluorescence (λ_(ex)=514 nm, λ_(em)=527nm) and OD₆₀₀ readings were taken using a Tecan Infinite Pro instrument.Fluorescence data were normalized to cell density by dividing by theOD₆₀₀ value.

Plasmid constructs Binding Name Class Res. Ori. Promoter Site GenepOHZif268-1 One-hybrid Carb SC101 P_(lac) (pIII-luc) −55 (Zif268)pIII-luxAB test plasmid P_(tet) (Zif268 fusion) Zif268 DBD-(M)- rpoZpOHZif268-2 One-hybrid Carb SC101 P_(lac)(pIII-luc) −55 (Zif268)pIII-luxAB test plasmid P_(tet) (Zif268 fusion) Zif268 DBD-(L)- rpoZpOHZif268-3 One-hybrid Carb SC101 P_(lac) (pIII-luc) −62 (Zif268)pIII-luxAB test plasmid P_(tet) (Zif268 fusion) Zif268 DBD-(M)- rpoZpOHZif268-4 One-hybrid Carb SC101 P_(lac) (pIII-luc) −62 (Zif268)pIII-luxAB test plasmid P_(tet) (Zif268 fusion) Zif268 DBD-(L)- rpoZpOHZif268-5 One-hybrid Carb SC101 P_(lac) (pIII-luc) −55 (Zif268)pIII-luxAB test plasmid P_(tet) (Zif268 fusion) rpoZ Zif268 DBDpOHZif268-6 One-hybrid Carb SC101 P_(lac) (pIII-luc) −55 (Zif268)pIII-luxAB test plasmid P_(tet) (Zif268 fusion) rpoZ L)-Zif268 DBDpOHZif268-7 One-hybrid Carb SC101 P_(lac) (pIII-luc) −62 (Zif268)pIII-luxAB test plasmid P_(tet) (Zif268 fusion) rpoZ Zif268 DBDpOHZif268-7: TTA One-hybrid Carb SC101 P_(lac) (pIII-luc) −62 pIII-luxABtest plasmid P_(tet) (Zif268 fusion) 5′GCGTTA rpoZ Zif268 DBD GCG3′pOHZif268-8 One-hybrid Carb SC101 P_(lac) (pIII-luc) −62 (Zif268)pIII-luxAB test plasmid P_(tet) (Zif268 fusion) rpoZ L)-Zif268 DBDpOHZif268-9 One-hybrid Carb SC101 P_(lac) (pIII-luc) −55 (Zif268)pIII-luxAB test plasmid P_(tet) (Zif268 fusion) rpoA Zif268 DBDpOHZif268-10 One-hybrid Carb SC10 P_(lac) (pIII-luc) −62 (Zif268)pIII-luxAB test plasmid P_(tet) (Zif268 fusion) rpoA Zif268 DBD SPZif268SP Kan F1 P_(gIII) — rpoZ-(M)-Zif268 DBD SPZif268-R24V SP Kan F1P_(gIII) — rpoZ-(M)-Zif268 DBD-R24V pAPZif268 AP Carb SC101 P_(lac) −62(Zif268) pIII-luxAB pAPZif268: TTA AP Carb SC101 P_(lac) −62 pIII-luxAB5′GCGTTA GCG3′

Genotypes of bacterial strains used Strain Genotype S1030 F′ proA+B+Δ(lacIZY) zzf::Tn10 lacI^(Q1) P_(N25)-tetR luxCDE/endA1 recA1 galE15galK16 nupG rpsL ΔlacIZYA araD139 Δ(ara, leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC) proBA::pir116 araE201 ΔrpoZ Δflu ΔcsgABCDEFG ΔpgaC λ⁻ S1059 F′proA+B+ Δ(lacIZY) zzf::Tn10 lacI^(Q1) P_(N25)-tetR luxCDE/endA1 recA1galE15 galK16 nupG rpsL ΔlacIZYA araD139 Δ(ara, leu)7697 mcrAΔ(mrr-hsdRMS- mcrBC) proBA::pir116 araE201 ΔrpoZλ⁻ pJC175e S1632 F′proA+B+ Δ(lacIZY) zzf::Tn10 lacI^(Q1) P_(N25)-tetR luxCDE/endA1 recA1galE15 galK16 nupG rpsL ΔlacIZYA araD139 Δ(ara, leu)7697 mcrAΔ(mrr-hsdRMS- mcrBC) proBA::pir116 araE201 ΔrpoZ Δflu ΔcsgABCDEFG ΔpgaCΔpspBC λ⁻ S2058 F′ proA+B+ Δ(lacIZY) zzf::Tn10 lacI^(Q1) P_(N25)-tetRluxCDE P_(psp) lacZ luxR P_(lux) groESL/endA1 recA1 galE15 galK16 nupGrpsL ΔlacIZYA araD139 Δ(ara, leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC)proBA::pir116 araE201 ΔrpoZ Δflu ΔcsgABCDEFG ΔpgaC λ⁻ S2059 F′ proA+B+Δ(lacIZY) zzf::Tn10 lacI^(Q1) P_(N25)-tetR luxCDE P_(psp)(T1) lacZ luxRP_(lux) groESL/endA1 recA1 galE15 galK16 nupG rpsL ΔlacIZYA araD139Δ(ara, leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC) proBA::pir116 araE201 ΔrpoZΔflu ΔcsgABCDEFG ΔpgaC λ⁻ S2060 F′ proA+B+ Δ(lacIZY) zzf::Tn10 lacI^(Q1)P_(N25)-tetR luxCDE P_(psp)(AR2) lacZ luxR P_(lux) groESL/endA1 recA1galE15 galK16 nupG rpsL ΔlacIZYA araD139 Δ(ara, leu)7697 mcrAΔ(mrr-hsdRMS-mcrBC) proBA::pir116 araE201 ΔrpoZ Δflu ΔcsgABCDEFG ΔpgaCλ⁻ S2208 F′ proA+B+ Δ(lacIZY) zzf::Tn10 lacI^(Q1) P_(N25)-tetR luxCDEP_(psp)(AR2) lacZ luxR P_(lux) groESL/endA1 recA1 galE15 galK16 nupGrpsL ΔlacIZYA araD139 Δ(ara, leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC)proBA::pir116 araE201 ΔrpoZ Δflu ΔcsgABCDEFG ΔpgaC λ⁻ pJC175e

DNA Sequence of Co-Zif268-DBD Fusion Protein.

Bases 997-1260 of m. musculus Zif268, corresponding the zinc fingerDNA-binding domain (residues 333-420, see Wolfe, S. A., Nekludova, L. &Pabo, C. O. DNA recognition by Cys2His2 zinc finger proteins. Annu RevBiophys Biomol Struct 29, 183-212 (2000)), were cloned downstream of theRNAP w subunit:

(SEQ ID NO: XX) atggcacgcgtaactgttcaggacgctgtagagaaaattggtaaccgttttgacctggtactggtcgccgcgcgtcgcgctcgtcagatgcaggtaggcggaaaggatccgctggtaccggaagaaaacgataaaaccactgtaatcgcgctgcgcgaaatcgaagaaggtctgatcaacaaccagatcctcgacgttcgcgaacgccaggaacagcaagagcaggaagccgctgaattacaagccgttaccgctattgctgaaggtcgtcgtgcggcgggcggcggcggcagcaccgcggcggctgaacgcccatatgcttgccctgtcgagtcctgcgatcgccgcttttctcgctcggatgagcttacccgccatatccgcatccacacaggccagaagcccttccagtgtcgaatctgcatgcgtaacttcagtcgtagtgaccaccttaccacccacatccgcacccacacaggcgagaagccttttgcctgtgacatttgtgggaggaagtttgccaggagtgatgaacgcaagaggcataccaaaatccatttaagacagaagtaa

Coding Sequence for Co-Zif268-DBD Fusion Protein.

The protein sequence of the ω-Zif268-DBD fusion protein is shown below.The residues highlighted in bold correspond to the w subunit, while theunderlined residues correspond to the 11-amino acid linker. Residuesdownstream of the linker comprise the Zif268-DBD (residues 333-420).

(SEQ ID NO: XX) M A R V T V Q D A V E K I G N R F D L V L V A A RR A R Q M Q V G G K D P L V P E E N D K T T V I AL R E I E E G L I N N Q I L D V R E R Q E Q Q E QE A A E L Q A V T A I A E G R R A A G G G G S T AA A E R P Y A C P V E S C D R R F S R S D E L T RH I R I H T G Q K P F Q C R I C M R N F S R S D HL T T H I R T H T G E K P F A C D I C G R K F A RS D E R K R H T K I H L R Q K

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All publications, patents and sequence database entries mentionedherein, including those items listed in the Summary, Brief Descriptionof the Drawings, Detailed Description, and Examples sections, are herebyincorporated by reference in their entirety as if each individualpublication or patent was specifically and individually indicated to beincorporated by reference. In case of conflict, the present application,including any definitions herein, will control.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above description, butrather is as set forth in the appended claims.

In the claims articles such as “a,” “an,” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention also includes embodiments in which more than one, or all ofthe group members are present in, employed in, or otherwise relevant toa given product or process.

Furthermore, it is to be understood that the invention encompasses allvariations, combinations, and permutations in which one or morelimitations, elements, clauses, descriptive terms, etc., from one ormore of the claims or from relevant portions of the description isintroduced into another claim. For example, any claim that is dependenton another claim can be modified to include one or more limitationsfound in any other claim that is dependent on the same base claim.Furthermore, where the claims recite a composition, it is to beunderstood that methods of using the composition for any of the purposesdisclosed herein are included, and methods of making the compositionaccording to any of the methods of making disclosed herein or othermethods known in the art are included, unless otherwise indicated orunless it would be evident to one of ordinary skill in the art that acontradiction or inconsistency would arise.

Where elements are presented as lists, e.g., in Markush group format, itis to be understood that each subgroup of the elements is alsodisclosed, and any element(s) can be removed from the group. It is alsonoted that the term “comprising” is intended to be open and permits theinclusion of additional elements or steps. It should be understood that,in general, where the invention, or aspects of the invention, is/arereferred to as comprising particular elements, features, steps, etc.,certain embodiments of the invention or aspects of the inventionconsist, or consist essentially of, such elements, features, steps, etc.For purposes of simplicity those embodiments have not been specificallyset forth in haec verba herein. Thus for each embodiment of theinvention that comprises one or more elements, features, steps, etc.,the invention also provides embodiments that consist or consistessentially of those elements, features, steps, etc.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and/or the understanding of one of ordinary skill in the art,values that are expressed as ranges can assume any specific value withinthe stated ranges in different embodiments of the invention, to thetenth of the unit of the lower limit of the range, unless the contextclearly dictates otherwise. It is also to be understood that unlessotherwise indicated or otherwise evident from the context and/or theunderstanding of one of ordinary skill in the art, values expressed asranges can assume any subrange within the given range, wherein theendpoints of the subrange are expressed to the same degree of accuracyas the tenth of the unit of the lower limit of the range.

In addition, it is to be understood that any particular embodiment ofthe present invention may be explicitly excluded from any one or more ofthe claims. Where ranges are given, any value within the range mayexplicitly be excluded from any one or more of the claims. Anyembodiment, element, feature, application, or aspect of the compositionsand/or methods of the invention, can be excluded from any one or moreclaims. For purposes of brevity, all of the embodiments in which one ormore elements, features, purposes, or aspects is excluded are not setforth explicitly herein.

1. A method for modulating the selection stringency duringviral-assisted evolution of a gene product, the method comprising: (a)introducing host cells into a lagoon, wherein the host cell comprises alow selection stringency plasmid and a high selection stringencyplasmid, wherein the low selection stringency plasmid comprises a viralgene required to package a selection viral vector into an infectiousviral particle, wherein at least one gene required to package theselection viral vector into an infectious viral particle is expressed inresponse to a concentration of a small molecule, and wherein the highselection stringency plasmid comprises a second copy of the viral generequired to package the selection viral vector into the infectious viralparticle, wherein at least one viral gene required to package theselection viral vector into an infectious viral particle is expressed inresponse to a desired activity property of a gene product encoded by thegene to be evolved or an evolution product thereof; (b) introducing aselection viral vector comprising a gene to be evolved into a flow ofhost cells through a lagoon, wherein the gene to be evolved produces anactive gene product or a weakly active or inactive gene product, whereinthe active gene product has an activity that drives the expression ofthe viral gene required to package the selection viral vector intoinfectious viral particles in the high selection stringency plasmid andwherein the weakly active or inactive gene product has a relativelylower activity than the activity of the active gene product; and (c)mutating the gene to be evolved within the flow of host cells, whereinthe host cells are introduced through the lagoon at a flow rate that isfaster than the replication rate of the host cells and slower than thereplication rate of the virus thereby permitting replication of theselection viral vector in the lagoon.
 2. The method of claim 1, furthercomprising isolating the selection viral vector comprising an evolvedproduct from the flow of cells and determining one or more properties ofthe evolved product.
 3. The method of claim 1, wherein the low selectionstringency plasmid contains a drift promoter that is activated by aconcentration of a small molecule inducer and/or prior viral infection.4. The method of claim 1, wherein the high selection stringency plasmidcontains a promoter that is activated by a desired property of a geneproduct encoded by the gene to be evolved or an evolution productthereof.
 5. The method of claim 4, wherein the low selection stringencyplasmid comprises a mutagenesis cassette under the control of asmall-molecule inducible promoter.
 6. The method of claim 5, wherein thelow selection stringency plasmid allows a high level of evolutionarydrift to occur when the drift promoter is activated in response to aconcentration of a small molecule inducer and/or prior viral infection.7. The method of claim 6, wherein the high selection stringency plasmidallows a low level of evolutionary drift to occur when the promoter isactivated in response to a desired activity property of a gene productencoded by the gene to be evolved or an evolution product thereof. 8.The method of claim 2, wherein the property of the gene to be evolvedoriginated from a weakly active or inactive starting gene.
 9. The methodof claim 1, wherein the high selection stringency comprises a T7promoter.
 10. The method of claim 1, wherein the low selectionstringency comprises a drift promoter that is activated by asmall-molecule inducer and/or prior viral infection.
 11. The method ofclaim 1, wherein the drift promoter is a P_(psp-tet) promoter.
 12. Themethod of claim 1, further comprising the use of negative selectionand/or positive selection.
 13. A method of using negative selectionduring viral-assisted evolution of a gene product, the methodcomprising: (a) introducing host cells into a lagoon, wherein the hostcell comprises a negative selection gene and a dominant negative mutantgene of a phage gene that decreases or abolishes packaging of theselection viral vector into infectious viral particles, wherein thedominant negative gene is expressed in response to an undesired activityof the gene to be evolved or an evolution product thereof or in responseto a concentration of a small molecule inducer; (b) introducing amixture of selection viral vectors into a flow of host cells through alagoon, wherein one type of selection viral vector comprises a negativeselection gene comprising an undesired gene or gene encoding anundesired property of the gene to be evolved and wherein another type ofselection viral vector comprises a positive selection gene comprising adesired gene or gene encoding a desired property of the gene to beevolved; and (c) mutating the gene to be evolved within the flow of hostcells, wherein the host cells are introduced through the lagoon at aflow rate that is faster than the replication rate of the host cells andslower than the replication rate of the virus thereby permittingreplication of the selection viral vector in the lagoon. 14-49.(canceled)
 50. A method of modulating the selection stringency and usingnegative selection during viral-assisted evolution of a gene product,the method comprising claim
 1. 51. (canceled)
 52. The method of claim 1,wherein the gene of interest to be evolved encodes a DNA-binding geneproduct. 53-54. (canceled)
 55. The method of claim 1, wherein expressionof the gene required to package the selection phagemid into infectiousparticles is driven by a promoter comprising a desired DNA binding sitefor the gene product.
 56. The method of claim 1, wherein expression ofthe dominant negative mutant of the phage gene that decreases orabolishes packaging of the selection phagemids into infectious phageparticles is driven by a promoter comprising an undesired DNA bindingsite for the gene product.
 57. The method of claim 1, wherein the hostcells are introduced into lagoons from a chemostat.
 58. The method ofclaim 1, wherein the host cells are prokaryotic cells amenable to phageinfection, replication, and production. 59-60. (canceled)
 61. The methodof claim 1, wherein a gene that is required to package the selectionviral vector into infectious viral particles is a gene that encodes forthe pIII protein.
 62. (canceled)
 63. The method of claim 1, wherein theselection viral vector comprises all viral genes required for thegeneration of viral particles, except for a full-length gene that isrequired to package the selection viral vector into infectious viralparticles. 64-68. (canceled)